Complete blood cell count (CBC) is one of the most valuable clinical tests to measure alternations in leukocyte numbers and to provide signs of infection and severe inflammation (e.g., sepsis). However, the CBC procedure is invasive, often requiring multiple blood samples for ex vivo laboratory analysis. Moreover, it does not provide dynamic cell information such as enhanced adhesiveness of leukocytes on the blood vessel walls, a cardinal feature of inflammation. We have developed a miniaturized oblique back-illumination microscope capable of noninvasive, high-resolution and label-free imaging of human microvasculature. The instrument was used to image and quantify circulating leukocytes, and the results are compared with CBC. We also investigated the leukocyte-endothelial interactions in inflamed conditions, revealing a drastic change in cell rolling speed. To provide clinicians with reliable and actionable results, we are developing a deep learning algorithm for automated quantification and movement analysis.
Sepsis is a life-threatening medical emergency caused by body's extreme inflammatory response to an infection. The important features of sepsis are the alternations in leukocyte numbers, enhanced adhesiveness of leukocytes on the blood vessel walls, and impaired microvasculature network that can ultimately lead to organ failure and death. To non-invasively quantify these parameters, we have built a miniaturized oblique back-illumination microscope capable of high-resolution and label-free imaging of human microvasculature in vivo. The developed instrument was used to capture high-speed videos of circulating, rolling, and adherent leukocytes. We have also developed a deep learning data analysis algorithm for rapid diagnosis of sepsis without drawing blood.
The present standard of blood cell analysis is an invasive procedure requiring the extraction of patient’s blood, followed by ex-vivo analysis using a flow cytometer or a hemocytometer. We are developing a noninvasive optical technique that alleviates the need for blood extraction. For in-vivo blood analysis we need a high speed, high resolution and high contrast label-free imaging technique. In this proceeding report, we reported a label-free method based on differential epi-detection of forward scattered light, a method inspired by Jerome Mertz's oblique back-illumination microscopy (OBM) (Ford et al, Nat. Meth. 9(12) 2012). The differential epi-detection of forward light gives phase contrast image at diffraction-limited resolution. Unlike reflection confocal microscopy (RCM), which detects only sharp refractive index variation and suffers from speckle noise, this technique is suitable for detection of subtle variation of refractive index in biological tissue and it provides the shape and the size of cells. A custom built high speed electronic detection circuit board produces a real-time differential signal which yields image contrast based on phase gradient in the sample. We recorded blood flow in-vivo at 17.2k lines per second in line scan mode, or 30 frames per second (full frame), or 120 frame per second (quarter frame) in frame scan mode. The image contrast and speed of line scan data recording show the potential of the system for noninvasive blood cell analysis.
Simultaneous imaging of cells expressing multiple fluorescent proteins (FPs) is of particular interest in applications such as mapping neural circuits, tracking multiple immune cell populations, etc. To visualize both in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissues, two-photon fluorescence microscopy (2PM) is a powerful tool that has found wide applications. However, simultaneous imaging of multiple FPs with 2PM is greatly hampered by the lack of proper ultrafast lasers offering multi-color femtosecond pulses, each targeting the two-photon absorption peak of a different FP. Here we demonstrate simultaneous two-photon fluorescence excitation of RFP, YFP, and CFP in human melanoma cells engineered to express a “rainbow” pallet of colors, using a novel fiber-based source with energetic, three-color femtosecond pulses. The three-color pulses, centered at 775 nm, 864 nm and 950 nm, are obtained through second harmonic generation of the 1550 nm pump laser and SHG of the solitons at 1728 nm and 1900 nm generated through soliton self-frequency shift (SSFS) of the pump laser in a large-mode-area (LMA) fiber. The resulting wavelengths are well matched to the two-photon absorption peaks of the three FPs for efficient excitation. Our results demonstrate that multi-color femtosecond pulse generation using SSFS and a turn-key, fiber-based femtosecond laser can fulfill the requirements for simultaneous imaging of multiple FPs in 2PM, opening new opportunities for a wide range of biological applications where non-invasive, high-resolution imaging of multiple fluorescent indicators is required.
Judith Runnels, Alicia Carlson, Costas Pitsillides, Brian Thompson, Juwell Wu, Joel Spencer, John Kohler, AbdelKareem Azab, Anne-Sophie Moreau, Scott Rodig, Andrew Kung, Kenneth Anderson, Irene Ghobrial, Charles Lin
Multiple myeloma (MM), the second most common hematological malignancy, initiates from a single site and spreads via circulation to multiple sites in the bone marrow (BM). Methods to track MM cells both in the BM and circulation would be useful for developing new therapeutic strategies to target MM cell spread. We describe the use of complementary optical techniques to track human MM cells expressing both bioluminescent and fluorescent reporters in a mouse xenograft model. Long-term tumor growth and response to therapy are monitored using bioluminescence imaging (BLI), while numbers of circulating tumor cells are detected by in-vivo flow cytometry. Intravital microscopy is used to detect early seeding of MM cells to the BM, as well as residual cancer cells that remain in the BM after the bulk of the tumor is eradicated following drug treatment. Thus, intravital microscopy provides a powerful, albeit invasive, means to study cellular processes in vivo at the very early stage of the disease process and at the very late stage of therapeutic intervention when the tumor burden is too small to be detected by other imaging methods.
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