Analyzing speckle contrast for HiLo microscopy optimization

Mazzaferri, Javier; Kunik, Darío; Bélisle, Jonathan M.; Singh, Kanwarpal; Lefrançois, Stéphane; Costantino, Santiago

doi:10.1364/oe.19.014508

Cited by 26 publications

(34 citation statements)

References 12 publications

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“…As expected from other work in the microscopic domain 18 , it was found that the speckle pattern for the structured illumination image needed to be coarser when imaging thicker samples (more than ~100 μm thick). In the ideal case, the transverse size of an imaged speckle grain was determined by the illumination NA 14,17 .…”

Section: Figure 1: Comparison Of Optical Sectioning Strengthsupporting

confidence: 76%

“…We have shown here a fast widefield optical sectioning method for the Mesolens using widefield camera image, no fine detail is lost 18 . This method presents a significant speed advantage over CLSM at comparable sectioning strength of ~5μm, being 30 times faster in raw data acquisition.…”

Section: Discussionmentioning

confidence: 88%

“…Although the plugin performed very well when we initially tested it with small datasets, it was very slow (~16 times slower than the current MATLAB implementation) when processing large Mesolens data and in some cases data could not be processed at all due to memory limitations (even when using a server for processing). It has been discussed in detail 18 that the strong out-of-focus background in thick samples presents a problem for HiLo imaging by reducing in-focus speckle contrast and hence making it more difficult to distinguish in-focus from out-of-focus regions in the image. To maintain high speckle contrast, a coarser speckle illumination pattern was used here.…”

Section: Discussionmentioning

confidence: 99%

“…The HiLo method can be implemented inexpensively by using a diffuser to create laser speckle with coherent (laser) light or with a grating and incoherent light. This diffuser-based method of generating laser speckle has been reported to be more robust than using a grating to study scattering samples 17 and the contrast of speckle can be easily adjusted to suit different samples 18 . SIM does provide super-resolution but it is not straightfoward to implement with the Mesolens: structured illumination would have to be introduced in the back aperture plane of the condenser because the back focal plane of the objective is not accessible in the current Mesolens design.…”

Section: Structured Illumination Microscopy (Sim) Is a Super-resolutimentioning

confidence: 99%

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Fast Optical Sectioning for Widefield Fluorescence Mesoscopy with the Mesolens based on HiLo Microscopy

Schniete

Franssen

Dempster

et al. 2018

Preprint

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We present here a fast optical sectioning method for optical mesoscopy based on HiLo microscopy, which makes possible imaging of specimens of up to 4.4 mm x 3 mm x 3 mm in volume in under 17 hours (estimated for a z-stack comprising 1000 images excluding computation time) with subcellular resolution throughout. Widefield epifluorescence imaging is performed with the Mesolens using a high pixel-number camera capable of sensor-shifting to generate a 259.5 Megapixel image, and we have developed custom software to perform HiLo processing of the very large datasets.Using this method, we obtain comparable sectioning strength to confocal laser scanning microscopy (CLSM), with sections as thin as 6.8±0.2 μm and raw acquisition speed of 1 minute per slice which is up to 30 times faster than CLSM on the full field of view (FOV) of the Mesolens of 4.4 mm with lateral resolution of 0.7 μm and axial resolution of 7 μm. We have applied this HiLo mesoscopy method to image fixed and fluorescently stained hippocampal neuronal specimens and a 5-day old zebrafish larva.

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Section: Figure 1: Comparison Of Optical Sectioning Strengthsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Structured Illumination Microscopy (Sim) Is a Super-resolutimentioning

confidence: 99%

See 2 more Smart Citations

Fast Optical Sectioning for Widefield Fluorescence Mesoscopy with the Mesolens based on HiLo Microscopy

Schniete

Franssen

Dempster

et al. 2018

Preprint

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“…Bottom: Deviations from uniformity in the images on top. This artifact in HiLo is a known consequence of statistical fluctuations in the total illumination dose, and is not corrected by increasing the exposure time 41 . Hadamard and stripe SIM microscopies avoided this artifact by providing illumination whose time-average intensity was precisely the same at all sample points.…”

Section: Discussionmentioning

confidence: 99%

Wide-area all-optical neurophysiology in acute brain slices

Farhi

Parot

Grama

et al. 2018

Preprint

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Optical tools for simultaneous perturbation and measurement of neural activity open the possibility of mapping neural function over wide areas of brain tissue. However, spectral overlap of actuators and reporters presents a challenge for their simultaneous use, and optical scattering and out-of-focus fluorescence in tissue degrade resolution. To minimize optical crosstalk, we combined an optimized variant (eTsChR) of the most blue-shifted channelrhodopsin reported to-date with a nuclear-localized red-shifted Ca 2+ indicator, H2B-jRGECO1a. To perform wide-area optically sectioned imaging in tissue, we designed a structured illumination technique that uses Hadamard matrices to encode spatial information. By combining these molecular and optical approaches we made wide-area maps, spanning cortex and striatum, of the effects of antiepileptic drugs on neural excitability and on the effects of AMPA and NMDA receptor blockers on functional connectivity. Together, these tools provide a powerful capability for wide-area mapping of neuronal excitability and functional connectivity in acute brain slices. ResultsA spectrally orthogonal Ca 2+ sensor and channelrhodopsin for 1-photon AON AON requires a spectrally orthogonal optogenetic actuator and activity reporter ( Fig. 1a). Examination of channelrhodopsin action spectra and Ca 2+ reporter excitation spectra suggested that the best approach for 1-photon AON was to use a blue-shifted channelrhodopsin and a red-shifted genetically encoded Ca 2+ indicator (RGECI). We thus set out to identify protein pairs suitable for this purpose.We began by comparing the single action potential responses of RGECIs in cultured neurons. jRGECO1a was the most sensitive (∆F/F = 54 ± 10%, n = ~120 neurons), followed by R-CaMP2 and jRCaMP1a, consistent with previous reports (Supplementary Fig. 1a-b, Supplementary Table 1) 15 . R-CaMP2 had the fastest kinetics (τon = 26 ± 10 ms, τoff = 270 ± 20 ms, n = ~120 neurons), followed by jRGECO1a (τon = 47 ± 1 ms, τoff = 440 ± 40 ms, n = ~120 neurons) and jRCAMP1a ( Supplementary Fig. 1ab, Supplementary Table 1). In HEK293T cells, under basal Ca 2+ conditions, jRGECO1a had the longest photobleaching time constant (τbleach = 81 ± 5 s, I561 = 44 W/cm 2 , n = 9 cells), followed by R-CaMP2 and jRCaMP1a ( Supplementary Table 1).Under typical imaging conditions (I561 = 0.1 W/cm 2 ), photobleaching of jRGECO1a was thus < 10% during 1 hr of continuous imaging. While photobleaching is often a concern for 1P imaging, these results established that this effect was minor for wide-area imaging of jRGECO1a. We selected jRGECO1a for its superior sensitivity and photostability.mApple-based fluorescent sensors, including jRGECO1a, are known to undergo photoswitching under blue light illumination 14,16 . We thus sought a blue-shifted channelrhodopsin that could drive spikes in jRGECO1a-expressing neurons at blue intensities low enough to avoid optical crosstalk. TsChR is the most blue-shifted published ChR (Fig. 1a), but was initially reported to produce only ~40% as much ph...

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In Vivo Intelligent Fluorescence Endo‐Microscopy by Varifocal Meta‐Device and Deep Learning

Chia,

Liao,

Vyas

et al. 2024

Advanced Science

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Endo‐microscopy is crucial for real‐time 3D visualization of internal tissues and subcellular structures. Conventional methods rely on axial movement of optical components for precise focus adjustment, limiting miniaturization and complicating procedures. Meta‐device, composed of artificial nanostructures, is an emerging optical flat device that can freely manipulate the phase and amplitude of light. Here, an intelligent fluorescence endo‐microscope is developed based on varifocal meta‐lens and deep learning (DL). The breakthrough enables in vivo 3D imaging of mouse brains, where varifocal meta‐lens focal length adjusts through relative rotation angle. The system offers key advantages such as invariant magnification, a large field‐of‐view, and optical sectioning at a maximum focal length tuning range of ≈2 mm with 3 µm lateral resolution. Using a DL network, image acquisition time and system complexity are significantly reduced, and in vivo high‐resolution brain images of detailed vessels and surrounding perivascular space are clearly observed within 0.1 s (≈50 times faster). The approach will benefit various surgical procedures, such as gastrointestinal biopsies, neural imaging, brain surgery, etc.

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Analyzing speckle contrast for HiLo microscopy optimization

Cited by 26 publications

References 12 publications

Fast Optical Sectioning for Widefield Fluorescence Mesoscopy with the Mesolens based on HiLo Microscopy

Fast Optical Sectioning for Widefield Fluorescence Mesoscopy with the Mesolens based on HiLo Microscopy

Wide-area all-optical neurophysiology in acute brain slices

In Vivo Intelligent Fluorescence Endo‐Microscopy by Varifocal Meta‐Device and Deep Learning

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