The illumination at the back focal plane is a circular annulus; it is shown as a point on one side of the optical axis for pictorial clarity only. Switching back and forth between epi and TIR can be done simply by placing or removing the opaque disk as shown. The size of the TIR fluorescence area on the sample increases with the angle of convergence of the rays at the back focal plane. In the case of TIR illumination, this is easily increased by expanding the beam width at the focusing lens just before the beam enters the microscope.
Arc illumination has the advantages of easy selection of excitation colors with filters and freedom from coherent light interference fringes, but it is somewhat dimmer because much of the arc lamp power directed toward the sample at subcritical angles is necessarily blocked. The vertical distances are exaggerated for clarity. The upper surface of the cell coverslip is put in optical contact with the prism lowered from above by a layer of immersion oil or glycerol.
The lateral position of the prism is fixed but the sample can be translated while still maintaining optical contact. The lower coverslip can be oversized and the Teflon spacer can be cut with gaps so that solutions can be changed by capillary action with entrance and exit ports. In configuration D, two incident beams split from the same laser intersect at the TIR surface, thereby setting up a striped interference pattern on the sample which is useful in studying surface diffusion Configuration E places the prism below the sample and depends on multiple internal reflections in the substrate.
However, only air or water immersion objectives may be used because oil at the substrate's lower surface will thwart the internal reflections. An extra lens just upbeam from the microscope base weakly focuses the TIR spot and permits adjustment of its position. If the objective has a long enough working distance, reasonable accessibility to micropipettes is possible. In an alternative approach for varying incidence angles over a continuous range, a hemispherical prism can be substituted for the trapezoidal prism The incident laser beam is directed along a radius line at an angle set by external optical elements.
TIRF for an upright microscope utilizing the integral optics in the microscope base and a trapezoidal prism on the condenser mount and movable up and down. The position of the beam is adjustable by moving the external lens. An alternative hemispherical prism configuration for variable incidence angle is also indicated to the left. Vertical distances are exaggerated for clarity.
Tissue culture dish plastic particularly convenient as a substrate in the upright microscope setup is also suitable, but tends to have a significant autofluorescence compared to ordinary glass. Also, the incidence angle should exceed the critical angle by at least a couple of degrees. Laser illumination produces interference fringes which are manifested as intensity variations over the sample area. For critical applications, it may be advisable to rapidly jiggle the beam e.
TIRF experiments often involve specially coated substrates. Derivatization generally involves pretreatment of the glass by an organosilane. A planar phospholipid coating possibly with incorporated proteins on glass can be used as a model of a biological membrane. Aluminum coating for surface fluorescence quenching can be accomplished in a standard vacuum evaporator; the amount of deposition can be made reproducible by completely evaporating a premeasured constant amount of aluminum.
After deposition, the upper surface of the aluminum film spontaneously oxidizes in air very rapidly. This aluminum oxide layer appears to have some similar chemical properties to the silicon dioxide of a glass surface; it can be derivatized by organosilanes in much the same manner. CM has the clear advantage in versatility; its method of optical sectioning works at any plane of the sample, not just at an interface between dissimilar refractive indices.
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Abstract Key events in cellular trafficking occur at the cell surface, and it is desirable to visualize these events without interference from other regions deeper within. Figure 1 Open in figure viewer PowerPoint. Figure 2 Open in figure viewer PowerPoint. Figure 3 Open in figure viewer PowerPoint.
Figure 4 Open in figure viewer PowerPoint. Intermediate layers In actual experiments in biophysics or cell biology, the interface may not be a simple interface between two media, but rather a stratified multilayer system. Qualitatively, several features can be noted: a Insertion of an intermediate layer never thwarts TIR, regardless of the intermediate layer's refractive index n 2. Figure 5 Open in figure viewer PowerPoint. Figure 6 Open in figure viewer PowerPoint. Figure 7 Open in figure viewer PowerPoint. Functionalized substrates TIRF experiments often involve specially coated substrates.
TIRF vs. Cell surface contacts illuminated by total internal reflection fluorescence. J Cell Biol ; 89 : — Google Scholar. Citing Literature. Volume 2 , Issue 11 November Pages Figures References Related Information. Close Figure Viewer. Browse All Figures Return to Figure. Previous Figure Next Figure. Email or Customer ID. Forgot password? Old Password. The functions of droplet formation, mixing, LSFM imaging and bypassing are sequentially integrated, from upstream to downstream.
Imaging is performed by the microscope objective from the side facet of the microfluidic device. C The device can be mounted on an inverted microscope for sustained image acquisition. Flow-based scanning eliminates the need of motorized stage and increases throughput. D The pictures of the actual droplets traveling through the microchannels. As seen before, flow-driven sectioning eliminates the need for mechanical scan. The chip offers high-throughput sample compartmentalization, manipulation, and volume imaging of microparticles, suggesting potential lab-on-a-chip applications such as embryo sorting and cell growth assays.
In their protocol, an additional processing step is required to produce optically-flat facets that allow clearer imaging through that side of the chip. Some earlier-described LSFM setups for standard sample mounting, such as inverted SPIM configurations, could also be considered as a possible solution to high-scattering side interfaces. Another microfluidic device of higher complexity has been designed for manipulating C. Apart from imaging and manipulation of C. An interesting microfluidic device for cell spheroid culture and analysis Patra et al.
The cells to be deposited in the squared microchambers could be harvested from a flow cytometer. The authors also propose a device fabricated such that the cultured spheroids can be imaged with SPIM, having a vertical and a horizontal objective. The combination of light-sheet fluorescence microscopy and microfluidic devices has shown, so far, three ways of combining both techniques:.
In great part, these fields of engineering rely strongly on custom-made systems that must be reproducible. Some designs have been successfully reported by different groups and used in meaningful and novel biology experiments, while others offer a proof-of-concept of the hardware that has been built. The most relevant design characteristics that have facilitated the combination of light-sheet illumination and microdevice technologies are highlighted in what follows, from both the viewpoint of the optical setup, and from that of microdevice fabrication.
Optical hardware research groups that have approached LSFM have created a variety of systems that offer different characteristics. Since a fluorescence imaging workstation can be a large investment for cell biology laboratories, selecting its appropriate benefits becomes paramount. Here, some of the capabilities that a system should offer for single-cell imaging and subcellular resolution are discussed. Related to sample sectioning, the use of ETLs over mechanical motion is preferred.
ETLs are a piece of optical equipment that can adjust their focus according to the current being applied, within a range limit. ETLs have very a fast response and allow for more compact setups with less mechanics. Additionally, the cost of an ETL is lower than that of a microstep piezoelectric stage.
Computational and Mathematical Methods in Medicine
Motor stages would still be required to place the sample in position, but the precision of such movements becomes particularly essential when these are driving the scan. There are several benefits associated to tunable lens over mechanical stages. First, ETLs offer increased scan speeds Fahrbach et al. As shown in Table 1 , not all methods can or have included tunable lenses in their setups, but they might offer other advantages which may suit a particular application.
Apart from temporal resolution, image quality and spatial resolution can be improved because the sample and the optical equipment remain static during sectioning see Mickoleit et al. Thus, the chance of blurring and misalignments decreases. Inertial forces during fast translations of a motor are avoided, which may unpredictably affect the adherence of the cells to the substrate or the flow of a fluid in a microchannel. There can also be space restrictions and limitations regarding sample mounting when the sample does not remain static and is translated back and forth from the detection objective for sectioning.
This requires enough space between the objective and the sample to avoid collisions. With respect to geometries, configurations that require a single objective have the advantage of having a single optical axis which facilitates their use in a standard microscope setup.
In the case of samples that remain static or are linearly scanned, an extra set of cylindrical lens in SPIM together with a mirror galvanometer motor can offer multidirectional views mSPIM by creating a pivoting light-sheet Huisken and Stainier, This type of pivoting has minor effects on the scan speed and in some cases may significantly reduce shadow artifacts. Regarding the imaging applications, most light-sheet systems covered above are suitable to image samples that range in size from single cells to small organisms.
Nevertheless, there are two exceptions: single-objective SPIM is restricted to sample sizes no larger than small cell cultures, while reflected LSFM can image all the way from small organisms down to sub-cellular structures and single molecules. For any microdevice, it is advisable to use materials along the optical path with homogeneous refractive indices and with the interfaces perpendicular to the path of light, avoiding irregular non-planar flow elements such as grooves, and circular pillars, barriers, or chambers.
The number of material interfaces should be kept minimal within the path of light because of the reflection and refraction effects. Also, when cutting PDMS microdevices the interfaces remain highly irregular. To combat outer-surface irregularities which interfere with the light-sheet, an additional step is required to either flatten the surface or couple the refractive indices with a refraction medium bath. Alternatively, Teflon and FEP — which have refractive indices close to those of water — can be used in building the microdevice. The main drawback of this non-mainstream fabrication technique is that it is not as straightforward as soft lithography with PDMS.
Another factor to keep in mind is accuracy and reproducibility in microfabrication. In all cases, the appropriate quality of microfabrication will be determined in great part by the desired application. Finally, keeping the thickness of the microdevice as fine as possible facilitates sample mounting when objectives of higher numerical aperture and magnification are used; these are prevalent in subcellular resolution imaging and have shorter working distances with which to operate.
The review has mostly focused on light-sheet cell-resolution imaging in microdevices, going through several setups that have successfully accomplished it. Other applications of LSFM in microfluidic devices, such as flow cytometry, are also detailed. The temporal and spatial resolution render LSFM ideal for the study of dynamic processes in cell biology, and microdevices can offer a finely-controlled platform for experimentation.
Thus, the combination of both emerging technologies is expected to grow, contributing to promising results in the area of cell motility and tracking; especially in the studying of essential processes like development, neurology, immune system, tissue repair, and tumor metastasis, all of which are of strong biomedical significance. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ancora, D. Phase-retrieved tomography enables mesoscopic imaging of opaque tumor spheroids.
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Recent Advances in Morphological Cell Image Analysis
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Accuracy and precision in quantitative fluorescence microscopy
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