Flow cytometry is an important technique in biomedical discovery for cell counting, cell sorting and biomarker detection. In vivo flow cytometers based on one-photon or two-photon excited fluorescence have been developed for more than a decade. One drawback of laser beam scanning two-photon flow cytometer is that two-photon excitation volume is very small due to short Rayleigh range of focused Gaussian beam. Hence, the sampling volume is much smaller than one-photon flow cytometer, which makes it challenging to count or detect rare circulating cells in vivo. Non-diffracting beam like Bessel beams and Airy beams have narrow intensity profiles with an effective spot size (FWHM) as small as several wavelengths, making them comparable to Gaussian beams. More significantly, the theoretical depth of field (propagation distance without diffraction) can be infinite, making them an ideal solution as a light source for scanning beam flow cytometry. The trade-off of using Airy beams rather than Gaussian beam is the fact that Airy beams have side lobes that contribute to background noise. Two-photon excitation can reduce this noise as the excitation efficiency is proportional to intensity squared. Therefore, we developed a two-photon flow cytometer using 2D Airy beams to form a light sheet that intersects the blood vessel. The set up can successfully detect and count flowing tumor cells in micro channel.
KEYWORDS: Signal to noise ratio, Particles, Point spread functions, Algorithm development, 3D image processing, Super resolution, Microscopy, Electroluminescence, Microscopes, Reconstruction algorithms
Choosing the localization algorithm in superresolution microscopy is an important factor in determining the resolution in such a modality. The point spread function (PSF) in defocused images has ring structures that can be used to localize the three-dimensional (3-D) position of single particles by calculating the ring center (x and y) and radius (z). As there is no well-developed mathematical model for a defocused PSF, it is difficult to perform a fitting-based algorithm in such images. A particle localization algorithm based on radial symmetry and ellipse fitting is developed to localize the centers and radii of defocused PSFs. Our method can localize the 3-D position of a fluorophore within 20-nm precision in three-dimensions in a range of 40 μm in z-dimension from defocused two-dimensional (2-D) images.
The temporal focusing two-photon microscope (TFM) is developed to perform depth resolved wide field fluorescence imaging by capturing frames sequentially. However, due to strong nonignorable noises and diffraction rings surrounding particles, further researches are extremely formidable without a precise particle localization technique. In this paper, we developed a fully-automated scheme to locate particles positions with high noise tolerance. Our scheme includes the following procedures: noise reduction using a hybrid Kalman filter method, particle segmentation based on a multiscale kernel graph cuts global and local segmentation algorithm, and a kinematic estimation based particle tracking method. Both isolated and partial-overlapped particles can be accurately identified with removal of unrelated pixels. Based on our quantitative analysis, 96.22% isolated particles and 84.19% partial-overlapped particles were successfully detected.
Two-photon fluorescence microscopy (TPFM) is a perfect optical imaging equipment to monitor the interaction between fast moving viruses and hosts. However, due to strong unavoidable background noises from the culture, videos obtained by this technique are too noisy to elaborate this fast infection process without video image processing. In this study, we developed a novel scheme to eliminate background noises, recover background bacteria images and improve video qualities. In our scheme, we modified and implemented the following methods for both host and virus videos: correlation method, round identification method, tree-structured nonlinear filters, Kalman filters, and cell tracking method. After these procedures, most of noises were eliminated and host images were recovered with their moving directions and speed highlighted in the videos. From the analysis of the processed videos, 93% bacteria and 98% viruses were correctly detected in each frame on average.
Flow cytometry is an important technique in biomedical discovery for cell counting, cell sorting and biomarker detection. In vivo flow cytometers, based on one-photon or two-photon excited fluorescence, have been developed for more than a decade. One drawback of laser beam scanning two-photon flow cytometer is that the two-photon excitation volume is fairly small due to the short Rayleigh range of a focused Gaussian beam. Hence, the sampling volume is much smaller than one-photon flow cytometry, which makes it challenging to count or detect rare circulating cells in vivo. Bessel beams have narrow intensity profiles with an effective spot size (FWHM) as small as several wavelengths, making them comparable to Gaussian beams. More significantly, the theoretical depth of field (propagation distance without diffraction) can be infinite, making it an ideal solution as a light source for scanning beam flow cytometry. The trade-off of using Bessel beams rather than a Gaussian beam is the fact that Bessel beams have small concentric side rings that contribute to background noise. Two-photon excitation can reduce this noise, as the excitation efficiency is proportional to intensity squared. Therefore, we developed a two-photon flow cytometer using scanned Bessel beams to form a light sheet that intersects the micro fluidic channel.
We use two-photon microscopy to monitor the infection process of marine zooplankton, Cafeteria roenbergensis (C.roenbergensis), by Cafeteria roenbergensis virus (CroV), a giant DNA virus named after its host. Here, we image C.roenbergensis in culture by two-photon excited NADH autofluorescence at video-rate (30 frame/s), and the movement of C.roenbergensis is recorded in live videos. Moreover, CroV is stained with DNA dye SYBR gold and recorded simultaneously with this two-photon microscope. We observed the initial infection moment with this method. The result demonstrates the potential use of two-photon microscopy to investigate the fast dynamic interaction between C.roenbergensis with virus CroV. After catching this initial moment, we will freeze the sample in liquid nitrogen for cryo-electron microscopy (EM) study to resolve the virus-host interaction at molecular level. The long-term goal is to study similar fast moving pathogen-host interaction process which could lead to important medical applications.
Intravital multiphoton microscopy has provided insightful information of the dynamic process of immune cells in vivo.
However, the use of exogenous labeling agents limits its applications. There is no method to perform functional imaging
of mast cells, a population of innate tissue-resident immune cells. Mast cells are widely recognized as the effector cells
in allergy. Recently their roles as immunoregulatory cells in certain innate and adaptive immune responses are being
actively investigated. Here we report in vivo mouse skin mast cells imaging with two-photon microscopy using
endogenous tryptophan as the fluorophore. We studied the following processes. 1) Mast cells degranulation, the first step
in the mast cell activation process in which the granules are released into peripheral tissue to trigger downstream
reactions. 2) Mast cell reconstitution, a procedure commonly used to study mast cells functioning by comparing the data
from wild type mice, mast cell-deficient mice, and mast-cell deficient mice reconstituted with bone marrow-derived mast
cells (BMMCs). Imaging the BMMCs engraftment in tissue reveals the mast cells development and the efficiency of
BMMCs reconstitution. We observed the reconstitution process for 6 weeks in the ear skin of mast cell-deficient Kit wsh/
w-sh mice by two-photon imaging. Our finding is the first instance of imaging mast cells in vivo with endogenous
contrast.
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