12 Cryomultiphoton imaging

: We describe multiphoton imaging with sample-temperature control to monitor animal cells and cells of intact plants during freezing, thawing and heating processes based on autoﬂuorescence intensity and lifetime. The sample temperature can be set with a heating and freezing stage to any value in the range between liquid nitrogen temperature (−196 °C; 77 K) and +600 °C (873 K) and changed with adjustable heating/freezing rates between 0.01 K/min and 150 K/min. Multiphoton imaging is realized with near-infrared femtosecond-laser excitation with different setups employing different laser sources. To illustrate the capabilities, imaging of animal cell samples with and without a cryoprotectant during freezing at cooling rates is presented. Lowering the temperature led to a signiﬁcant increase of the intracellular ﬂuorescence intensity and modiﬁcations. Fluorescence lifetime imaging indicated an increase of the mean lifetime with decreasing temperature. Furthermore, to illustrate imaging of plant samples, Arabidopsis thaliana leaves were employed. The measurements revealed thermally-induced changes of ﬂuorescence lifetime and intensity as well as morphological alterations in the distribution of chloroplasts. The measurements illustrate the general usefulness of multiphoton imaging to investigate freezing and thawing effects on animal and plant cells even at temperatures commonly used for cryopreservation.


Introduction
The basis for cryopreservation, nowadays a routine technique for long-term preservation, is the fact that basically all enzymatic or chemical activities inside biological material stop at low enough temperatures [1].A variety of human and animal cells, tissues such as cornea, skin, pancreatic tissue, liver slices, and heart valves as well as plant cells, sprouts, seeds, and even whole plants can be stored in this way for long time periods [2] with the possibility to recover the original function of the material provided that hazards like ice formation, which may damage cellular structures, are limited.Most freezing methods aim to prevent ice formation inside cells by applying suitable freezing conditions through the control of the freezing rate and/or the addition of cryoprotectants, i.e., substances that protect biological tissue from freezing damage, or by dehydration of the material prior to freezing [3].In fact, cryopreservation methods range from very slow cooling to instant vitrification of a sample by placing it directly in liquid nitrogen [4,5].The success of the different methods is related to microscopic processes which are induced by the freezing of extra-and intracellular liquids [4].To be able to monitor a sample during the freezing process on-line on a microscopic scale could be helpful to optimize freezing protocols and to better understand fundamental temperature-induced processes [6].Both are scientifically as well as economically significant due to the general importance of cryopreservation.With this background in mind, we have combined multiphoton imaging systems with precise sample temperature control.This combination provides multiphoton imaging capability based on fluorescence intensity, lifetime and spectral characteristics of externally applied or intrinsic fluorophores during freezing/thawing/heating processes with subcellular resolution.The temperature control in the range from liquid nitrogen temperatures up to +600 °C is realized with a heating/freezing microscope stage.To illustrate the imaging capabilities, mammal cells were imaged at different temperatures with and without the addition of a cryoprotectant as well as parts of plant leaves.
Microscopic imaging of cooled or frozen samples can in principle also be realized with a conventional optical microscope combined with a cryostage.However, this limits the application to the observation of surfaces or very thin samples.In contrast, multiphoton imaging, which relies on near-infrared excitation, focusing with high NA microscope objectives, and a laser-scanning setup provides intrinsic 3D-imaging capability with high penetration depth, i.e., also below the surface inside a sample, due to low near-infrared absorption and scattering rates in biological material as well as further well-known advantages like low out-of-focus phototoxicity [7][8][9].The method is an established microscopy technique which has already been extensively used to imagine human/animal/plant materials with a variety of objectives ranging from basic research to clinical diagnosis of diseases by in vivo imaging [8][9][10][11][12][13][14] and can be adapted for precise material processing as well [15].

Heating and cooling stage
The heating and cooling stage (MDS 600, Linkham, "cryostage") controls the sample temperature in the range between −196 °C and +600 °C, i.e., between 77 K and 873 K, respectively.A photograph of the stage is shown in Fig. 12.1 [16].The sample is placed onto a metal block (labelled "11" in Fig. 12.1) whose temperature is continuously measured and precisely controlled.The user can set desired values for temperature and cooling/heating rate in a control software interface.The rate can be set to zero, i.e., the temperature is kept fixed, or to any value in the range between ±0.1 K/min and ±150 K/min.Based on this input, control electronics dynamically regulate the amount of electrical heating and/or the flow rate of liquid-nitrogen through the stage.
For imaging, different kinds of microscope objectives can be combined with the cryostage.Dry objectives, with large working distances, provide the possibility to close the interior of the stage with a sealed glass window above the sample which improves the thermal isolation of the sample.In Fig. 12.1, the stage is shown with this cover in place.Nevertheless, also high NA oil-immersion objectives can be used.High NA ob- x-y motor control cable, 6: cooling water connection for housing (inlet valve), 7: cooling water connection for housing (outlet valve), 8: temperature control cable, 9: nitrogen gas (outlet), 10: nitrogen exhaust for flushing (inlet) of stage housing, 11: metal block where sample is placed, 12: illumination aperture.The interior of the stage is covered with a removable lid with a glass window for thermal shielding.The outer dimensions of the stage housing are 94 mm × 149 mm.From [16].
jectives are advantageous for two-photon imaging because with the same mean laser power a higher in-focus intensity can be realized which results in higher two-photon signals.When using oil-immersion objectives, the interior of the cryostage with the sample holder cannot be sealed but still be somewhat closed with a cooling jacket which fits around the objective to provide some thermal isolation.Nevertheless, in this case during cooling/heating, the oil still provides thermal contact between the temperature-controlled sample and the objective which is at ambient temperature.This may lead to a temperature gradient between sample surface and bottom, and some uncertainty of the precise sample temperature in particular for very low temperatures.Throughout this chapter all temperatures values given relate to the values measured inside the metal block onto which the sample was placed.We found that oil-immersion objectives can still be used down to −80 °C, although the immersion oil (Zeiss immersion oil 518F) solidifies at temperatures below −50 °C.In addition to the possibility to regulate the temperature, the stage provides the option to place the sample in a small dish which can be moved in x and y directions by microstep motors with a resolution of 0.05 µm and a maximum travel range of 15 mm in either direction.This option in principle allows centering a specific region of the sample in the focus of the objective or mapping a larger region of interest with mosaic imaging by stitching together individual images from neighboring regions.

Multiphoton imaging
The basis for multiphoton fluorescence imaging is multiphoton absorption, i.e., the simultaneous absorption of two or more photons by a molecule which results in an energetic excitation that corresponds to the sum of the energies of the photons involved [7,17].Molecules with one-photon-absorption bands in the UV or blue-visible wavelength region can, in this way, be excited by simultaneous absorption of two (or more) low energy photons in the near-infrared wavelength region.However, such a multiphoton absorption occurs at a significant rate only at high enough light intensities (MW/cm 2 to GW/cm 2 ) [7].Those can be realized inside the focal volume (in the range of a femtoliter [11]) when ultrashort laser pulses are focused with high NA objectives.Outside the focal volume, the intensity is then not sufficient for multiphoton absorption to occur.This provides intrinsic 3D imaging capability with high resolution.For image acquisition, the laser focus is raster scanned across the sample.Fluorescence is collected (in case of reflection geometry) by the same focusing optics, separated from the incoming laser light by a dichroic mirror and a short-pass filter, which further discriminates residual laser light, and then detected for each pixel.Besides fluorescence, also other nonlinear signals like second-harmonic generation (SHG) can arise from the interaction between the intense laser light in the focal region and the sample depending on the specific sample properties as well as excitation conditions and can be used for imaging as well [17].
In order to elucidate the possibility to non-invasively image a section from the inside of a frozen sample, i.e., below the surface with the two-photon cryomicroscope, horizontal sections of an ice block containing fluorescent microspheres are shown in Fig. 12.2 [16].The microspheres can be clearly identified against the nonfluorescent ice.Imaging up to a depth of 1.5 mm limited only by the working distance of the objective was possible, however, a higher laser power was necessary for imaging inside ice compared to imaging inside liquid water [16].

Imaging systems
The cryostage has been combined with several multiphoton imaging systems (2PM Cryo [16], DermaInspect [18], MPTflex [14]; all JenLab GmbH) that are all based on the well-known laser-scanning microscope setup with samples kept fixed, scanning of the laser focus, and signal detection in reflection geometry (Fig. 12.3 (a)) [8].The platform 2PM Cryo (Fig. 12.3 (b)) which was used for the animal cell imaging comprises of the cryostage (Fig. 12.1) in combination with a modified optical microscope (Axioskop, Zeiss).For cell imaging, the 2PM Cryo was outfitted with a high NA objective (Zeiss, 40×, NA 1.3) which provided a maximum field of view of 250 µm × 250 µm and image resolutions of 0.5 µm in lateral and 1-2 µm in axial directions, respectively.Other objectives (oil immersion or dry) could in principle also be used for imaging.The multiphoton cryomicroscope 2PM Cryo can be combined with different excitation lasers.For the measurements presented here, either a Ti:sapphire laser (Vitesse 800, Coherent; 800 nm center wavelength, 100 fs pulse width, 80 MHz repetition rate) or the frequency-doubled output of a fiber laser (mfiber, MenloSystems, 780 nm center wavelength, 250 fs pulse width, 250 MHz repetition rate) was used [19].
Alternatively, the cryostage can be combined with the clinical multiphoton tomographs DermaInspect and MPTflex (JenLab GmbH), respectively [11,18].These systems typically incorporate an fs laser (MaiTai XF, Newport Spectra Physics) which provides 100 fs pulses at a repetition rate of 80 MHz.The center wavelength of the pulses is tunable in the near-infrared wavelength range from 710 nm to 920 nm.The DermaInspect was used for imaging of the Arabidosis thaliania samples (Section 12.3.2) with the laser wavelength set at 800 nm and a maximum mean laser power of a few tens of mW [20].
In a further experimental setup, the cryostage was combined with a mobile multiphoton-imaging system (MPTflex, JenLab GmbH; Fig. 12.3 (c)).The MPTflex includes an articulated mirror arm for beam delivery and a mechanical arm that allows to freely position the scan-detector head such that otherwise hard-to-reach sample areas become accessible [21].Depending on the configuration, the MPTflex also features coherent anti-Stokes Raman scattering (CARS) [22,23] and fluorescence lifetime imaging (FLIM) capability [24].To realize the FLIM capability, time-correlated singlephoton counting (TCSPC) with a temporal resolution of about 170 ps (Becker & Hickl, SPC card 830) was employed in the system used here.FLIM images were obtained by pseudo-color coding the mean fluorescence decay times which were obtained from pixelwise fitting a two-exponential decay to the fluorescence data [24,25].The mean fluorescence decay time τ m = a 1 τ 1 + a 2 τ 2 was calculated from the decay time fitting parameters τ 1 , τ 2 and the associated normalized amplitudes a 1,2 .Intensity and FLIM images were typically obtained without further averaging at imaging speeds of 13.4 s per frame with 512 × 512 pixels and 256 × 256 pixels, respectively.Spectra were measured with a fiber-coupled thermoelectrically-cooled CCD-array spectrometer (B & W Tek, BTC112).The spectrometer had a wavelength-dependent resolution of a few nm.During the spectral measurements, the laser focus was continuously scanned over the sample and the signals summed for 30 s.To increase the signal-to-noise ratio, all spectra were smoothed by 30-point adjacent averaging.The spectra were not corrected for the spectral transmission of the system and detector sensitivity.

CHO cells
As a model for animal cells, Chinese hamster ovary K1 cells (CHO-K1 cells ECACC, Sigma Aldrich #07K011) grown in a monolayer were imaged.Before starting the imaging procedure, the fluorescent growth medium was rinsed off the samples with phosphate buffered saline (PBS).The cells were placed on microscope cover glasses which were positioned on the temperature-controlled metal block of the cryostage (Fig. 12.1).
The cryoprotectant dimethyl sulphoxide (DMSO) [3] was added to some of the cell samples up to a concentration of 10 vol.% to investigate its effect on the freezing process.

Plant samples
For imaging plant cells, small parts (≈ 2 × 2 mm 2 ) of leaves of Arabidopsis thaliana (ecotype Colombia -0) were cut and fixed between two microscope coverslips which were then placed inside the cryostage on top of the temperature-controlled metal block (Fig. 12.1).No additional cleaning or treatments of the samples were performed.Cooling/heating rates of 10 K/min were used in between the measurements.Furthermore, a leaf of Dieffenbachia, a popular house plant purchased at a local store, was imaged without further preparation or treatment before the measurement.A part of one of the leaves (few cm 2 ) was fixed on the temperature-controlled metal block inside the cryostage and cooled while the rest of the plant was at ambient temperature.During the imaging procedure, the leaf was not detached from the rest of the plant.The images were recorded a few minutes after the metal block reached the set temperature to allow the imaged region to reach a temperature equilibrium.Only a small part (≈ 200 µm × 200 µm) of the cooled region was imaged.

Cell monolayer imaging
Cooling a cell that is inside a solution or in tissue below the freezing point leads initially to ice formation inside the extracellular environment because the cell membrane prevents (at first) intracellular ice formation.In this case, the supercooled intracellular water remains at a higher chemical potential than the partially frozen extracellular solution.This leads to a thermodynamic nonequilibrium which causes, depending on the cooling rate, subsequent cellular dehydration or intracellular ice formation [26][27][28].With a slow cooling rate, the intracellular liquid can flow out of the cell through the semipermeable membrane and join the extracellular matrix before solidifying.This results in changes of the pH value as well as the ion concentrations inside the extracellular region and may cause the tertiary structure of proteins to fold, effectively eliminating the original cell properties [29,30].Furthermore, severe damage to cells may result from mechanical interactions with extracellular ice crystals [1].Intracellular ice formation threatens intracellular structures and the cell membrane [31].The balance between the water permeability of the membrane and the rate of intracellular ice formation leads to different optimal cooling rates for different cell types.For cryopreservation, typically, a slow freezing with cooling rates between 0.3 K/min and 10 K/min with 5-10 % cryoprotective agent in the medium is beneficial.An alternative approach is to immediately place the sample with a high cryoprotective agent concentration between 40 % and 70 % into liquid nitrogen (vitrification) [6].This however may kill many cells.The blue dots are measurement data, the red line a two exponential fit, (e) fit results of the data shown in (d).Laser parameters: wavelength: 780 nm; laser power: 13 mW.From [52].
An example for FLIM imaging of animal cells (CHO-K1 cells) is shown in Fig. 12.4.
Starting at room temperature, the temperature was set to 0 °C with a freezing rate of 10 K/min.In the fluorescence intensity (Fig. 12.4 (a)) and FLIM (Fig. 12.4 (b)) images, the nonfluorescent cell nuclei appear as dark round regions inside the cells.With a laser wavelength of 780 nm, which was employed here, NAD(P)H molecules are efficiently excited by two-photon absorption molecules and constitute the main source of the fluorescence signals [18].NAD(P)H is abundant inside the cell cytoplasm as well as inside mitochondria.The intracellular space outside the cells is presumably filled with water and/or ice, does not contain fluorescent material, and appears black.The fluorescent decay data were fitted with two-exponential decay functions.Comparing the FLIM images (Fig. 12.4 and 12.5) reveals an increase of bluish colored pixels which indicates a general increase of the τ 2 values with decreasing temperature.This can also be seen in the change of the distribution of the τ 2 -decay times (compare Fig. 12.4 (c) and 12.5 (c)), which is shifted to higher values.The characteristics of the representative decay curve in Fig. 12.5 (d) changed to a single-exponential decay.These temperature-induced changes reflect the temperature-dependent characteristics of the NAD(P)H molecules which can also be observed when freezing pure NADH in vitro as described in [32].The effect of the cryoprotectant DMSO and the influence of different cooling rates on cell samples are illustrated in Fig. 12.6 and 12.7.The sample was cooled with a rate of 10 K/min starting at room temperature and imaged at 25 °C, −40 °C and −80 °C.During the time taken to record an imaging, the temperature was kept constant.As can be seen (Fig. 12.6 and 12.7), the fluorescence intensity increases with deceasing temperature and in addition the cell morphology as well as the fluorescence pattern change significantly.The most intense fluorescence stems from the cell membranes.In the sample with 10 % DMSO the cell shape morphology is also affected by the temperature.Fig. 12.7 shows autofluorescence intensity images at the same temperatures as in Fig. 12.6 of samples cooled with a higher cooling rate of 100 K/min.In Fig. 12.7, the cell morphology appears to be less affected than the morphology of the cells that underwent the slower freezing procedure.The same strong increase of fluorescence intensity with decreasing temperature is nevertheless observable.

In situ imaging of plants
In general, plants contain quite a few intrinsic fluorophores that, in combination, generate fluorescence in the range from the UV to the far-red spectral region.These fluorophores are molecules and substances like chlorophyll a and b, ferulic acid, NADPH, FAD and rubisco [33].Chlorophyll, which is present inside the chloroplasts of green mesophyll cells of leaves, generates fluorescence in the red (maximum around 680 nm) and far-red (maximum around 740 nm) spectral range [34,35] whereas fluorescence emitted by ferulic acids, which are bound to cell walls, is maximum near 440 nm and 520 nm [33,34,36,37].Fluorescence imaging is an important tool to study plant conditions.The technique has been applied to investigate spatial and tempo- ral changes due to stress put on plants, environmental effects, herbicide resistance, and pathogen toxin responses on a microscopic level [37][38][39][40][41][42][43][44] as well as to monitor photosynthetic chlorophyll activity [35,45,46].The cryostage in combination with a multiphoton imaging system can be used to monitor temperature-induced effects in plants.As an example, fluorescence from an Arabidopsis thaliana leaf was recorded at temperatures of 0 °C and −20 °C (Fig. 12.8 (a) and (b)).The sample was cooled, starting from room temperature at a rate of 10 K/min.The fluorescence was excited by two-photon absorption with a laser power of 8 mW at 800 nm.The images show horizontal "optical cuts" of the same region of the mesophyll cell layer.Chloroplasts inside a cell can be seen in high detail (Fig. 12.8 (a)); a second cell is partly visible at the bottom part of the figure.The chloroplasts appear due to the strong chlorophyll fluorescence as bright oval objects with sizes of a few µm and are aligned along the cell walls.Further structures and shapes between the chloroplasts appear less bright and more diffuse.Besides fluorescence intensity, fluorescence lifetime can also be recorded and depicted in pseudo-color coded FLIM images.The corresponding FLIM image (Fig. 12.8 (c)) based on pseudo-color coding of the mean decay time τ m indicates two different main sources of fluorescence.Pixels with mean decay times below and above 0.8 ns are colored in red and green, respec-tively.By this coloring, it becomes clear that the regions with short decay times contain chloroplasts which appear in red and the regions with longer decay times are the surrounding more diffuse structures which appear in green.The chloroplasts contain chlorophyll that emits strong and short-lived fluorescence signals.Therefore, mean decay times below 0.8 ns can be attributed to chlorophyll, decay times around and above 1 ns to fluorescence of molecules bound to cell walls, in particular ferulic acids, whose fluorescence is typically emitted at shorter wavelengths and decays slower than chlorophyll fluorescence [20,34,35,37].The FLIM images were obtained from twoexponential fitting.Two-and even three-exponential decays of the fluorescence have been identified [47,48] in several studies employing both one-and two-photon excitation [44,49,50].Excitation: 800 nm pulses from Ti:sapphire laser.From [53].
red region in the image with fast fluorescence decays.The histograms of the mean decay time (Fig. 12.8 (e)) show a striking change from 0 °C (peaks below 0.5 ns, and around 1.0 ns) to −20 °C (maximum around 0.5 ns and strong maximum around 1 ns).Spectra of the fluorescence from the imaged region (Fig. 12.8 (f)) indicate signals in the range between 400 and 600 nm stemming from cell wall fluorescence, and additional signals above 600 nm as a result of the chlorophyll fluoresce [34,36].Further temperature effects as well as additional local maxima are present in the spectra which reflect the temperature influence on chlorophyll, and cell wall fluorescence and indicate an even more complex situation.A more detailed discussion of these temperature effects and the participating fluorescent molecules as well as their temperature-dependent dynamics is, however, beyond the scope of this chapter.
The cryostage in combination with multiphoton imaging can also be used to image parts of leaves which are still attached to the rest of the plant.To illustrate the possibility to investigate temperature-induced effects on fluorescence intensity and lifetime on a plant leaf, the cryostage was combined with the multiphoton tomograph MPTflex, which due to its articulated arm and freely positionable scan-detector head offers easier accessibility to large samples than the microscope platform.The leaf was pressed onto the cooling block of the cryostage and the scan-detector head placed onto the sample from above.Fig. 12.9 shows three FLIM images of the same leaf region after cooling of the cryostage to set temperatures of −25 °C, −50 °C and −80 °C, respectively.The chloroplasts containing chlorophyll as the main fluorophore exhibit shorter decay times than the other intracellular regions inside the cells which appear in green and blue pseudo-colors.The intracellular fluorescence results from other fluorophores like ferulic acids [36] which exhibit longer fluorescence lifetimes than chlorophyll [51].The chloroplasts appear mainly red in Fig. 12.9 (a).With decreasing temperature (Fig. 12.9 (b) and (c)), more green and blue false-colored pixels appear indicating longer decay times.This is similar to the observations described before with Arabidopsis thaliana leafs (Fig. 12.8) [51] including probably temperature-induced structural changes in the imaged region, i.e., chloroplast relocations which are indicated by the arrows in Fig. 12.9 (a) and (b).Also, a movement of the whole sample resulting from the cooling is recognizable.
The images of animal cell and plant samples discussed above were based on detecting fluorescence light.Therefore, water and ice, which are nonfluorescent, were not directly visible.However, the actual forming of ice and its distribution inside a sample is of particular interest because ice formation is a major cause of cell damage.In principle, water and ice as well as different ice structures can be detected by their specific Raman transitions [27].While Raman imaging is very time consuming, nonlinear Raman processes like CARS [29] could be a worthwhile additional imaging modality for multiphoton cryoimaging as already realized in a recent version of the MPTflex which incorporates additional CARS imaging capability [29].Imaging is possible in regions inside the cooled as well as uncooled parts.Adapted from [52].

Conclusion
We have described temperature-dependent multiphoton imaging of fluorescence intensity and lifetime of animal and plant cell samples.The sample temperature could be controlled by a stage in the range between −196 °C and +600 °C with cooling/heating rates from 0.1 K/min to 150 K/min by balancing the amounts of liquid nitrogen cooling and electrical heating.Multiphoton imaging was realized by nearinfrared femtosecond-laser pulse excitation and laser imaging scanning systems (2PM Cryo, MPTflex, DermaInspect) in reflection geometry.For the animal cells, an increase of fluorescence intensity and lifetime with decreasing temperature was observed.Influences of a cryoprotectant (DMSO) on the freezing process could be clearly seen to affect the cell morphology.In an in vitro measurement, parts of plant leaves were imaged; "in vivo" results were presented from a leaf that was still attached to the plant during the imaging procedure.For the plant samples, temperature-induced changes like chloroplast relocation and changes in the fluorescence decay characteristics were observed as well as a trend to larger decay values with decreasing temperature in line with properties of chlorophyll reported in the literature.

Fig. 12 . 1 :
Fig. 12.1: Cooling and heating stage Linkham MDS 600 viewed from the top.1, 2: micro-step motors for x-y positioning, 3: tube for liquid nitrogen in-flow, 4: nitrogen exhaust gas for housing flushing (outlet), 5:x-y motor control cable, 6: cooling water connection for housing (inlet valve), 7: cooling water connection for housing (outlet valve), 8: temperature control cable, 9: nitrogen gas (outlet), 10: nitrogen exhaust for flushing (inlet) of stage housing, 11: metal block where sample is placed, 12: illumination aperture.The interior of the stage is covered with a removable lid with a glass window for thermal shielding.The outer dimensions of the stage housing are 94 mm × 149 mm.From[16].

Fig. 12 . 2 :
Fig. 12.2: Optical horizontal two-photon sections through an ice block which contains fluorescent microspheres.Indicated is the imaging depth inside the ice below the surface.The images show sections of 60 × 60 µm 2 .From [16].

Fig. 12 . 3 :
Fig. 12.3: (a) Scheme of multiphoton imaging systems based on laser scanning setup and signal detection in reflection geometry.(b) Imaging system 2PM Cryo (without laser).The cryostage is combined with a laser scanning microscope.Next to the microscope are the control electronics for the stage and a Dewar with liquid nitrogen.(c) The cryostage can also be combined with the mobile multiphoton imaging system MPTflex.The MPTflex is a multipurpose multiphoton imaging system which incorporates fluorescence intensity, lifetime, SHG, and CARS imaging capability and features a scan-detector head attached to an articulated optical arm.

Fig. 12 . 4 :
Fig. 12.4: FLIM measurement of CHO cells at a temperature of 0 °C.(a) Two-photon fluorescence image, (b) FLIM image (fluorescence decay time τ 2 color-coded according to color distribution shown in (c), (c) decay time distribution of the FLIM image shown in (b) and color code for τ 2 decay time values, (d) fluorescence decay curve at the position at the crossing of the blue lines in (a) and (b).The blue dots are measurement data, the red line a two exponential fit, (e) fit results of the data shown in (d).Laser parameters: wavelength: 780 nm; laser power: 13 mW.From[52].

Fig. 12 .
4 (d) and 12.4 (e) exemplary show decay time data with the fitting curve and the corresponding fit parameters.The distribution of the values of the longer fluorescence decay time τ 2 , shown in Fig. 12.4 (c), exhibits a maximum around 1000 ps.Fig. 12.5 shows the situation after further cooling to a set temperature of −100 °C at a rate of 10 K/min.Again, fluorescence intensity and FLIM images are shown in the figure.

Fig. 12 . 5 :
Fig. 12.5: FLIM measurement of CHO cells at a temperature of −100 °C.(a) Two-photon fluorescence image, (b) FLIM image (fluorescence decay time τ 2 color-coded according to color distribution shown in (c), (c) mean decay time distribution of the FLIM image shown in (b) and color code for τ 2 decay time values, (d) fluorescence decay curve at the position at the crossing of the blue lines in (a) and (b).The blue dots are measurement data, the red line a two exponential fit, (e) fit results of the data shown in (d).Laser parameters: wavelength: 780 nm; laser power: 13 mW.From [52].

Fig. 12 . 8 :
Fig. 12.8: (a)-(d) Multiphoton imaging of a plant leaf (Arabidopsis thaliana).(a) and (b) fluorescence intensity images, (c) and (d) color-coded mean fluorescence lifetime (FLIM) images, red: pixels with decay time below 0.8 ns, green: pixels with decay time above 0.8 ns.The color code is illustrated in (e) which shows the decay time distributions.(f) Micro-spectra collected from the imaged regions.Excitation: 800 nm pulses from Ti:sapphire laser.From[53].

Fig. 12 . 9 :
Fig. 12.9:In vivo FLIM images of a plant leaf.The plant with the imaged region was cooled by the cooling block of the cryostage while the rest of the plant was still at room temperature.Set temperatures: (a) −25 °C, (b) −50 °C, (c) −80 °C.The pseudo colors encode mean fluorescence decay times according to the color legend.The arrows point in each image to the same region where temperature-induced structural changes are visible.Laser parameters -wavelength: 820 nm; laser power: 13 mW.(d) During the measurement the leaf was not detached from the living plant.Imaging is possible in regions inside the cooled as well as uncooled parts.Adapted from[52].