Ratio Imaging. Dividing one image by another to detect changing conditions. Images collected at different times, wavelengths, polarities, etc

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1 Ratio Imaging Dividing one image by another to detect changing conditions Images collected at different times, wavelengths, polarities, etc Most common use of ratio imaging is to provide a quick spectral fingerprint

2 Ratio Imaging 458 excitation (ph-insensitive) stomach 514 excitation (ph-sensitive) Ratio image (calculation) ph4 perfusate + dye ph3 70 µm

3 Ratio Imaging What are the best wavelengths to choose for ratioing? Dynamic range of ratio values A: 9-fold change B (isosbestic point): no change C: 2-fold change A B C Reality of your noisy detector and specimen may mean that too extreme a change gives poor data

4 Ratio Imaging Ratios help compensate for Loss of dye (photobleach/leak) Variability in local dye concentration Variability in cell thickness (non-confocal) Fluctuations in excitation light intensity Changes in detector sensitivity/gain Ratios will NOT help compensate for Saturation in detector or fluorophore response Incorrectly adjusted gains/settings on imaging device Contribution of background autofluorescence Nothing gets around the garbage in garbage out rule

5 Probes Ion selective probes K+ PBFO, PBFI Na+ SBFO, SBFI Ca++ Fura2, Indo1, calcium cameleon FP. Fluo3, Fura Green, FuraRed, calcein ph BCECF, SNARFs, SNAFL, all FPs Mg++ Mag-Fura2 Cl- SPQ, MQAE, YFP HCO 3 -? Membrane potential 8-ANEPPS, oxynols, styrl dyes, DiOC 7 Volume Calcein Sensors must change response over a relevant physiological range Sensors must be selective for the event of interest

6 Probes Fura2-acetoxymethylester (AM) 1-10 µm AM fluorescence Toxic cleavage products Partially cleaved forms Buffering Fura2-AM esterases µm Fura2 Cell homeostasis perturbation Cell survival mm SPQ Hypotonic shock µm SPQ

7 Collect images with minimal time separation Camera detector: entire image1, change settings, entire image2 PMT based confocal: emission ratio multiple images simultaneously essential for processes on msec scale excitation ratio frame switching (same as camera) line switching (1-10 ms betw. colors) point switching? Speed of your imaging device (and your perfusion system) must be compatible with the timing of the biological process you want to capture

8 Quantitative Imaging Set detector background correctly: Background image pixels all greater than zero #pixels #pixels Gray level

9 Quantitative Imaging Minimal noise is that your values will vary ±1 gray level ~ 10% noise ~1% noise 10 ± 1 10 ± 1 ~ 20% noise 100 ± ± 1 ~ 2% noise Ratio value and images are noisier than raw data High gray values (more significant digits) for best precision

10 Quantitative Imaging Test linearity of response: Doubling light to detector should give double the signal Fluorescence microscope: neutral density filter Laser scanning microscope: reflectance, AOTF control No saturated signals: Detector Digital image dynamic range Fluorophore saturation

11 Quantitative Imaging 150 Cl-NERF 514nm/458nm intensity ratio x ph Even with optimized, linear responses of excitation and emission, you must always work within range of dye response

12 Quantitative Imaging Calculating Ratios Threshold: eliminate low values from image that would give noisy ratio values. ONLY if most data is clearly separated from noise floor Subtract background (autofluorescence + detector) Extract average values from regions of interest in fluorescence images, then manually ratio for highest precision Calculate ratio image for presentation

13 Quantitative Imaging Daily calibration Never assume the system is the same as yesterday Calibrate at the end of every experiment Suggestion: single point ratio calibration (dye in known solution) so you can scale response to known calibration curve Gold standard Ratio 5 Today ph 4 ratio = /10 Ratio Scaled calibration curve ph ph

14 Thick specimen imaging Light scatter Light absorption More trouble to get light in, get light out and still believe you know what your numbers mean. Increasing depth of focus means checking the fidelity of your light path even before you get a biological specimen on the stage!

15 Picking an objective for an aqueous environment Zeiss 63X NA 1.4 oil 640nm 580nm Normalized fluorescence Leitz 50X NA 1.0 water Normalized fluorescence Depth of focus into SNARF-1 ocean (µm)

16 Picking an objective for an aqueous environment SNARF-1 emission ratio µm µm µm ph 8.0 Step 1 All AOK in solution Step 2 How check if it will work in thick specimens like native tissue samples?

17 An inert control: Lucifer Yellow Broad excitation Broad emission Boring EX EM

18 An inert control: Lucifer Yellow ph and ion insensitive Cheap Small: 450 g/mole Dextran conjugates Use to check fidelity of optical path Ideal condition is to match excitation and emission colors with your real probe 640nm/580nm ratio SNARF-1 Lucifer yellow ph

19 The live tissue nemesis: Light Scatter LUCIFER YELLOW fluorescence (normalized) 640nm 580nm Depth of focus into tissue (µm)

20 Does ratio imaging correct for light scatter artifacts in tissue? Lucifer Yellow fluorescence ratio (640nm/580nm) Crypt lumen Adjacent tissue Depth of focus into tissue (µm)

21 Where am I? Confocal reflectance (rat gastric surface) pits lumen mucus epithelium 50 µm XZ plane XY plane