Modular platform for low-light microscopy

Kim, Tae Jin; Tuerkcan, Silvan; Ceballos, Andrew; Pratx, Guillem

doi:10.1364/boe.6.004585

Cited by 17 publications

(28 citation statements)

References 30 publications

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“…The pattern of binding of [ 18 F]HFB, consisting of preferential binding to fragmented membranes of non-viable cells, has not been observed with other PET radiolabels. Previously, radioluminescence microscopy has been applied to imaging of 9-[4-[ 18 F]fluoro-3-(hydroxymethyl)-butyl]guanine ([ 18 F]FHBG) uptake in HeLa cells expressing herpes simplex type 1 virus (HSV1) thymidine kinase [29], binding of [ 64 Cu]rituximab in single Ramos B lymphocytes [39], uptake of [ 18 F]FDG [30,40] and 3′-deoxy-3′-[ 18 F-]fluorothymidine ([ 18 F]FLT) [41] in MDA-MB-231 cells. In these studies, binding of the radiolabel was only observed in viable cells, with minimal background, ruling out the possibility of a technical artefact.…”

Section: Discussionmentioning

confidence: 99%

Single-Cell Imaging Using Radioluminescence Microscopy Reveals Unexpected Binding Target for [18F]HFB

et al. 2017

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Purpose Cell-based therapies are showing great promise for a variety of diseases, but remain hindered by the limited information available regarding the biological fate, migration routes and differentiation patterns of infused cells in trials. Previous studies have demonstrated the feasibility of using positron emission tomography (PET) to track single cells utilising an approach known as positron emission particle tracking (PEPT). The radiolabel hexadecyl-4-[18F] fluorobenzoate ([18F]HFB) was identified as a promising candidate for PEPT, due to its efficient and long-lasting labelling capabilities. The purpose of this work was to characterise the labelling efficiency of [18F]HFB in vitro at the single-cell level prior to in vivo studies. Procedures The binding efficiency of [18F]HFB to MDA-MB-231 and Jurkat cells was verified in vitro using bulk gamma counting. The measurements were subsequently repeated in single cells using a new method known as radioluminescence microscopy (RLM) and binding of the radiolabel to the single cells was correlated with various fluorescent dyes. Results Similar to previous reports, bulk cell labelling was significantly higher with [18F]HFB (18.75 ± 2.47 dpm/cell) than [18F]FDG (7.59 ± 0.73 dpm/cell). However, single-cell imaging using RLM revealed that [18F]HFB accumulation in live cells (8.35 ± 1.48 cpm/cell, n = 9) was not significantly higher than background levels (4.83 ± 0.52 cpm/cell, n = 12) and was 1.7 fold lower than [18F]FDG uptake in the same cell line (14.09 ± 1.90 cpm/cell, n = 13; p < 0.01). Instead, [18F]HFB was found to bind significantly to fragmented membranes associated with dead cell nuclei, suggesting an alternative binding target for [18F]HFB. Conclusion This study demonstrates that bulk analysis alone does not always accurately portray the labelling efficiency, highlighting the need for more routine screening of radiolabels using RLM to identify heterogeneity at the single-cell level.

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Section: Discussionmentioning

confidence: 99%

Single-Cell Imaging Using Radioluminescence Microscopy Reveals Unexpected Binding Target for [18F]HFB

et al. 2017

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“…Recently, we developed a “low-light microscope” (LLM) to offer a cost-effective and modular alternative to commercial bioluminescence microscopes 18 . This system is entirely constructed from commercial lenses and optomechanical parts.…”

Section: Introductionmentioning

confidence: 99%

“…The design of the microscope enables a level of performance comparable to commercial bioluminescence microscopes (e.g. LV200), except for a minor reduction in field-of-view (∼30% area) due to the smaller physical aperture of the 4× objective used as tube lens 18 . Since the microscope is highly modular, the setup can be tailored to the researcher's needs, and additional capabilities such as microfluidics, intravital imaging or motorized X-Y stage can be added 19 .…”

Section: Introductionmentioning

confidence: 99%

“…This is important because (1) the boundary of the radioactivity is not clearly defined on the radioluminescence image, and (2) ROIs of different sizes receive variable contribution from the image background. ROIs should be placed only on cells that are spaced far enough from one another to avoid crosstalk 18 . Once the ROIs have been placed, the number of recorded decays can be tallied and converted into units of number of molecules initially present inside a single cell,…”

Section: Introductionmentioning

confidence: 99%

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Modular low-light microscope for imaging cellular bioluminescence and radioluminescence

2017

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Low-light microscopy methods are receiving increased attention as new applications have emerged. One such application is to allow longitudinal imaging of light-sensitive cells with no phototoxicity and no photobleaching of fluorescent biomarkers. Another application is for imaging signals that are inherently dim and undetectable using standard microscopy, such as bioluminescence, chemiluminescence, or radioluminescence. In this protocol, we provide instructions on how to build a modular low-light microscope (1-4 d) by coupling two microscope objective lenses, back-to-back from each other, using standard optomechanical components. We also provide directions on how to image dim signals such as radioluminescence (1-1.5 h), bioluminescence (∼30 min) and low-excitation fluorescence (∼15 min). In particular, radioluminescence microscopy is explained in detail as it is a newly developed technique, which enables the study of small molecule transport (eg. radiolabeled drugs, metabolic precursors, and nuclear medicine contrast agents) by single cells without perturbing endogenous biochemical processes. In this imaging technique, a scintillator crystal (eg. CdWO4) is placed in close proximity to the radiolabeled cells, where it converts the radioactive decays into optical flashes detectable using a sensitive camera. Using the image reconstruction toolkit provided in this protocol, the flashes can be reconstructed to yield high-resolution image of the radiotracer distribution. With appropriate timing, the three aforementioned imaging modalities may be performed altogether on a population of live cells, allowing the user to perform parallel functional studies of cell heterogeneity at the single-cell level.

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“…Given the challenging nature of imaging radionuclides at the single‐cell level with high sensitivity, the performance of RLM in terms of spatial resolution and sensitivity must be as high as possible. Currently, the design of the microscope (eg, the parameters of the microscopy objective, scintillator and camera) has been optimized empirically, through trial and error . For instance, a 10‐μm thin Lu 2 O 3 scintillator has been used in some experiments to enhance spatial resolution and sensitivity.…”

Section: Introductionmentioning

confidence: 99%

In silico optimization of radioluminescence microscopy

Wang

Sengupta

Kim

et al. 2017

Journal of Biophotonics

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Radioluminescence microscopy (RLM) is a high-resolution method for imaging radionuclide uptake in live cells within a fluorescence microscopy environment. Although RLM currently provides sufficient spatial resolution and sensitivity for cell imaging, it has not been systematically optimized. This study seeks to optimize the parameters of the system by computational simulation using a combination of numerical models for the system's various components: Monte-Carlo simulation for radiation transport, 3D optical point-spread function for the microscope, and stochastic photosensor model for the electron multiplying charge coupled device (EMCCD) camera. The relationship between key parameters and performance metrics relevant to image quality is examined. Results show that Lu O :Eu yields the best performance among 5 different scintillator materials, and a thickness: 8 μm can best balance spatial resolution and sensitivity. For this configuration, a spatial resolution of ~20 μm and sensitivity of 40% can be achieved for all 3 magnifications investigated, provided that the user adjusts pixel binning and electron multiplying (EM) gain accordingly. Hence the primary consideration for selecting the magnification should be the desired field of view and magnification for concurrent optical microscopy studies. In conclusion, this study estimates the optimal imaging performance achievable with RLM and promotes further development for more robust imaging of cellular processes using radiotracers.

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Modular platform for low-light microscopy

Cited by 17 publications

References 30 publications

Single-Cell Imaging Using Radioluminescence Microscopy Reveals Unexpected Binding Target for [18F]HFB

Single-Cell Imaging Using Radioluminescence Microscopy Reveals Unexpected Binding Target for [18F]HFB

Modular low-light microscope for imaging cellular bioluminescence and radioluminescence

In silico optimization of radioluminescence microscopy

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