Simultaneous spatiotemporal computational super-resolution and multi-parametric �uorescence microscopy

We describe here a protocol for performing simultaneous spatiotemporal computational super-resolution and multi-parametric �uorescence microscopy. Our approach does not need specialized instrumentation, utilizes a GPU for faster data evaluation, and produces mutually consistent structure and dynamics data.


Introduction
We describe a protocol to perform simultaneous multi-parametric analysis using fast and sensitive cameras (EMCCD and sCMOS) and a combination of super-resolution and spectroscopy techniquessuper resolution radial uctuation (SRRF)/super-resolution optical uctuation imaging (SOFI)/deconvolution, uorescence correlation spectroscopy (FCS) analysis, number and brightness analysis (N&B), and FCS diffusion law analysis.
SRRF [1] (with its roots in SOFI [2]) analyzes the uctuations in radiality stacks to obtain super resolved images.One could also apply deconvolution algorithms to the raw image stack [3].Imaging uorescence correlation spectroscopy (Imaging FCS) [4] [5] analyzes the uctuations in uorescence intensity in a pixel to estimate the diffusion coe cient (D) and the number of particles in that pixel (N).The relationship between observation area and diffusion coe cient is determined in FCS diffusion law analysis to understand the mode of diffusion [6].The concentration and brightness at each pixel is estimated from the mean and variance of intensity in a pixel in N&B analysis [7] [8].
We introduce a GPU-supported, camera-based approach to achieve high spatiotemporal resolution from a single data set and perform multiple analyses on it to extract maximum information.Reagents 1.For the point spread function (PSF) calibration of the system, a sample exhibiting free diffusion is needed.We recommend DOPC (2-dioleoyl-sn-glycero-3-phosphocholine; Avanti Polar Lipids, Alabama, USA) bilayer containing a uorescent label.Examples of uorescent labels include: • Lipilight 488 (Idylle, Paris, France) for 488 nm excitation.
• With the extra 2× magni cation, the image pixel size for the iXon EM + 860 EMCCD and Sona 4.2B-11 sCMOS is 120 nm and 65 nm, respectively.6. On-stage temperature and CO 2 incubator (Chamlide TC, Live Cell Instrument, South Korea).• Use the magni cation slider if higher magni cation is required.
• Use the dual-emission image splitter if dual channel measurements are to be done.
2. Excite the sample with suitable laser powers and record a stack of images at high temporal resolution.
As an example, we use the following settings for our measurements: • Laser powers of 100-300 µW for 488 nm excitation and 0.1-1 mW for 561 nm excitation when using an EMCCD with EM gain of 300.If sCMOS is used as detector, higher laser powers in mW range may be necessary.
3. Record at 1,000 frames per second (fps) for bilayer measurements and 500 fps for cell membrane measurements.If su cient signal-to-noise ratio (SNR) is achievable, faster recording rates can be used.
• In Andor Solis, for the EMCCD, the kinetic mode of image acquisition was used and the 'baseline clamp' was always used to minimize the baseline uctuation.The camera was operated using 10 MHz pixel readout speed.Maximum analog-to-digital gain was set to 4.7, and 0.45 µs vertical shift speed was used.
The EM gain used was 300.
• Similarly, for the sCMOS, the pixel readout rate used was 200 MHz in an overlap readout mode.The rolling electronic shuttering mode and 12-bit dynamic range was used.
4. Save the image stack as a 16-bit tiff image stack since that is the input for the ImFCS plugin in ImageJ.

B. Data pre-processing
To maintain a good SNR, post-acquisition spatial or temporal binning might be necessary.We suggest the following based on our experience: a. Imaging FCS -1×1 binning or 2×2 binning depending on SNR.For EMCCD, 1×1 binning usually works at 240 nm image pixel size.If higher magni cation or sCMOS is used, 2×2 binning might be required.b.FCS diffusion law -The plot can usually be generated and t with spatial binning of 1×1 to 5×5.But at low SNR, the smaller bins have large errors and higher spatial binnings might be required.c.N&B analysis -Time bin to at least 10 ms per frame, otherwise the brightness calibration might underestimate the uorescence probability of the uorescent protein.We use a higher time binning of 20 ms to get better differentiation of the background and the cell for intensity thresholding, d.Computational super-resolution microscopy -We observed optimal results for SOFI/SRRF after time binning to 200 ms per frame.For deconvolution, temporally bin all the frames to obtain a single timeaveraged image.
C. Calibration of point spread function (PSF) for Imaging FCS 1. calibrate the PSF for the experimental setup, load the bilayer measurement le into the ImFCS plugin in ImageJ.
2. Set the appropriate values for following parameters in the plugin: frame time, correlator (p, q), pixel size, NA, and λ 1 .2. Set the appropriate values for following parameters in the plugin: frame time, correlator (p, q), pixel size, NA, λ 1 and bleach correction (refer to ImFCS manual for details).

The program will compute and displayed a plot of the diffusion coe cient values for various combinations of PSF and binning values
3. Calculate and t the ACFs with a suitable diffusion model.To exclude outliers or non-converged ts, threshold the data using the available lters (e.g.N or D lter).
4. In the "Diff.Law" tab, the diffusion law plot can be generated and t with a straight line for square binning of desired range.

7 .
Appropriate emission dichroic mirror and optical lters.8. Image processing software a. Fiji-ImageJ • For Imaging FCS, FCS diffusion law and N&B -Imaging FCS (ImFCS) 1.52 plugin included in ImageJ update site.Also can be downloaded here.The ImFCS 1.52 manual is available here.• For SOFI and SRRF -NanoJ-SRRF plugin (version 1.14Stable1) included in ImageJ update site.b.For deconvolution -Huygens Professional version 20.04 (Scienti c Volume Imaging, The Netherlands)9.An NVIDIA graphics processing unit (GPU) for faster analyses.

Procedure A. Data acquisition 1 .
Using the appropriate microscopy setup and camera. .

4 .
Refer to the ImFCS manual for more details.D. Data analyses Refer to ImFCS, NanoJ-SRRF and Huygens manuals for more details.a. FCS and FCS diffusion law 1.Load the image stacks in ImFCS 1.52 plugin.