What is confocal microscopyused for

Michael graduated from the University of Salford with a Ph.D. in Biochemistry in 2023, and has keen research interests towards nanotechnology and its application to biological systems. Michael has written on a wide range of science communication and news topics within the life sciences and related fields since 2019, and engages extensively with current developments in journal publications.

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Laser scanningconfocal microscopy

Croix, C. M., Shand, S. H. & Watkins, S. C. (2018) Confocal microscopy: comparisons, applications, and problems. BioTechniques, 39(6S). doi: 10.2144/000112089 https://www.future-science.com/doi/full/10.2144/000112089

The image captured is generally in the range of several hundred nanometers, and so large samples are scanned to allow a larger image to be stitched together later. Adjusting the focal point of the laser and microscope along each of the three axes allows scientists to build up a three-dimensional image of the sample of interest.

Greenwood, Michael. 2021. What is Confocal Fluorescence Microscopy?. News-Medical, viewed 24 November 2024, https://www.news-medical.net/life-sciences/What-is-Confocal-Fluorescence-Microscopy.aspx.

Lavrentovich, O. D. (2012) Confocal Fluorescence Microscopy. Optical Imaging and Spectroscopy. doi: 10.1002/0471266965 https://onlinelibrary.wiley.com/doi/full/10.1002/0471266965.com127

Measurement:On the day of measurement, the cell supernatant was discarded, and the cells were washed 2 x for 5 min in 200 µL FluroBrite medium with 5% FBS, 2 mM glutamine and 1% pen/strep. Plates including 200 µL medium/well were  transferred to the VANTAstar plate reader and GFP and mcherry fluorescence was determined either with matrix scan or spiral averaging using the bottom optic setting to evaluate the simulated transfection efficiency. Afterwards cells were fixated in 4% PFA and stained with Hoechst 33342 for 15 min, washed 3 times in PBS, and read again with the VANTAstar using matrix scan or spiral averaging. With the measurement of the Hoechst signal, total cell counts were determined as internal standard for the evaluation of the transfection efficiency.

In practical application, fluorophores are selected by the desired excitation and emission wavelengths, the intensity of returned light (quantum yield), and the fluorescence time. Other considerations that may encourage the use of a particular fluorophore over another include the molecular weight, as larger molecules may be sterically hindered in some applications, the presence of other fluorophores that may provide overlapping signals, and the specificity of the fluorescent label.

Samples frequently undergo fixation before microscopy to carefully preserve the structural features of the sample. Formaldehyde has historically been used for this purpose, rapidly penetrating cell membranes and forming disulfide bridges between cysteine residues in proteins, preserving even the fine structural details of antigen sites.

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Is confocal microscopylightmicroscopy

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In cases where proteins need not be preserved, when examining the presence of small molecules for example, alcohol fixation at low temperatures may instead be used. In order to ensure complete penetration of the fixation agent, the cells are exposed to mild detergents, increasing the permeability of the cell membrane.

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Confocal microscopyprinciple

However, the availability and diversity of laser types accommodated by laser scanning confocal microscopy mean that it remains the most popular option.

The lower limit of detection for the simulated transfection efficiency in 20,000 cells per well was calculated based on the SD of the blank (= 100% WT HeLa w/o GFP+/mcherry+-HeLas) and the slope of the respective standard curve. The VANTAstar is able to reliably detect a transfection efficiency down to 5.3% measuring GFP fluorescence (fig. 4). The detectable transfection efficiency could be improved even further down to 3.1% by measuring mcherry fluorescence. This benefit can be mainly attributed to the reduced presence of cellular autofluorescence as well as of autofluorescing cell culture medium components in the red wavelength range.

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Unwanted visual noise from the sample, besides light from the spot in focus with the laser, is omitted from the final image by the use of a pinhole aperture that prevents light scattered from higher angles from passing through. The pinhole is placed along the same plane as the sample to ensure that only photons traveling in a direct line from the sample to the microscope are detected. This is said to be in a conjugate focal plane, hence the portmanteau “confocal”.

Confocal microscopyppt

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Greenwood, Michael. (2021, January 22). What is Confocal Fluorescence Microscopy?. News-Medical. Retrieved on November 24, 2024 from https://www.news-medical.net/life-sciences/What-is-Confocal-Fluorescence-Microscopy.aspx.

Cells are transfected with exogenous DNA to study the regulation of gene and protein expression. To monitor transfection efficiency, a reporter gene is often attached to the gene of interest to monitor its insertion into the cell’s genome. Fluorescent proteins like Green Fluorescent Protein (GFP) are often used as such reporters during transfection efficiency experiments. The reporter gene can either be present on the same vector as the gene of interest or can alternatively be located on a separate plasmid. The success of a transfection experiment is defined by the ratio of cells expressing the used reporter which “reports” on the insertion of the gene of interest. This ratio is also known as transfection efficiency.

Confocal microscopyPDF

Antibodies are the most common targeting agent in fluorescent labeling due to their versatility and specificity. Specificity can be improved further by a process known as blocking, flooding the sample with a protein cocktail that occupies non-specific binding sites and exhausts the protein-crosslinking ability of any remaining formaldehyde.

Some energy is thought to be consumed by non-radiative decay processes, and the difference in energy between the incident and emitted photon is known as Stokes shift. As the system relaxes vibrationally the electron returns to the ground state, releasing the remaining difference between the electron energy levels as a photon. The decay rate generally follows first-order kinetics, with most commonly employed fluorophores emitting within nanoseconds.

Experimental ProcedureCell culture:GFP+/mcherry+- and WT-HeLa cells were precultured separately in tissue culture flasks in DMEM with 10% FBS, 2 mM glutamine and 1% pen/strep. On the day before the measurement, cells were detached and seeded with 20,000 cells/well on a 96-well plate with µclear bottom in 200 µL medium. GFP+/mcherry+-HeLas were seeded in ratios from 0%-100% by mixing them with WT-HeLa cells to simulate transfection efficiency to different extents. Cells were allowed to attach to the cell culture surface overnight.

In confocal microscopy a laser beam is focused to a specific depth within a sample, and similarly to ordinary light microscopes, any reflected or emitted light is detected by a properly positioned microscope.

As discussed, fluorophores are selected for compatibility with the sample under investigation and favorable spectral properties. The great sensitivity of fluorescent probes means that only a comparatively small number need be present in the sample to achieve a sufficient signal.

Greenwood, Michael. "What is Confocal Fluorescence Microscopy?". News-Medical. https://www.news-medical.net/life-sciences/What-is-Confocal-Fluorescence-Microscopy.aspx. (accessed November 24, 2024).

Molecular “lock and key” mechanisms can be exploited to ensure that fluorophores bond with structures of interest, potentially only activating or deactivating once in position. Antibodies form highly specific bonds with their target structure and are frequently linked to fluorophores in order to track the number and distribution of the target in situ. More recently, peptide and nucleic acid sequences have been employed to similar effect in identifying cellular components.

Confocal microscopydiagram

Sanderson, M. J., Smith, I., Parker, I. & Bootman, M. D. (2016) Fluorescence Microscopy. Cold Spring Harbor Protocols, 10. doi: 10.1101/pdb.top071795 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711767/

Additionally, significantly lower intensities of laser light may be employed to obtain a sufficient signal, reducing the risks to the sample associated with photon bombardment. Other sub-types of confocal microscope are available, depending on the application, which limits exposure to photons and improves the collection time of images by using a slit or spinning disc in place of a pinhole.

Smith, C. L. (2011). Basic Confocal Microscopy. Current Protocols in Neuroscience. doi: 10.1002/0471142301.ns0202s56  currentprotocols.onlinelibrary.wiley.com/.../0471142727.mb1411s81

If cells are stably transfected with the genetic blueprint for fluorescent proteins, they will consistently express these reporters. Thereupon, a fluorescence microscope or a fluorescence microplate reader can be used to detect them. By mixing fluorescent with wild type (WT) cells, transfection efficiency can be simulated to different extents (fig.1). Here, HeLa cells were transfected with the genetic sequences for GFP and mcherry and mixed in increasing ratios with WT-HeLas without fluorescent reporter to simulate transfection efficiency.

Greenwood, Michael. "What is Confocal Fluorescence Microscopy?". News-Medical. 24 November 2024. .

Over- or under-populating a sample with fluorophores can be disadvantageous, generating a noisy signal or incomplete structural elucidation, and so care must be taken in selecting an appropriate concentration.

Confocal fluorescence microscopy is a commonly used optical imaging method in biology, combining fluorescence imaging with confocal microscopy for increased optical resolution. This article will discuss the principles of fluorescence and confocal microscopes, and describe the stages of fluorophore selection and sample preparation.

The repeated activation of fluorophores eventually induces a phenomenon known as photobleaching, leaving the molecule completely unable to fluoresce. In time-sensitive studies, this must be accounted for, as the number of emitted photons will decline following repeated bouts of excitation.

Many naturally occurring compounds are intrinsically fluorescent, and a vast library of additional specially engineered fluorophores is in constant development. Organic fluorophores frequently contain a number of double bonds and polyaromatic structures that provide delocalized electrons throughout the molecule, which are then capable of excitation.

BMG LABTECH plate readers reliably detect cells expressing a fluorescent marker down to ~600 cells/well in a 96-well plate and thereby represent a valuable alternative to microscopes to monitor transfection efficiency. Both matrix scan and spiral averaging deliver accurate results, allowing the user to choose between speed and image-like resolution. The readers also reliably detect transfection efficiency experiments based on red-shifted dyes. These come with the advantage of avoiding most of the autofluorescence derived primarily from media and cell-derived components.

Depending on the transparency of the sample, and the wavelength of exciting light, samples can be imaged to a depth of a few hundred micrometers. Maintained scanning over a period of time provides time-lapsed images that allow scientists to observe the dynamics of tagged molecules or structures over time.

Nwaneshiudu, A., Kuschal, C., Sakamoto, F. H., Anderson, R. R., Schwarzenberger, K. & Young, R. C. (2012) Introduction to confocal microscopy. Journal of Investigative Dermatology, 123(12), pp.1-5. ohsu.pure.elsevier.com/.../introduction-to-confocal-microscopy

Disadvantages ofconfocal microscopy

Fixation, by definition, is unsuitable in dynamic microscopy applications that intend to observe the function of the cell or tissue over time. Live-cell imaging raises a number of issues with regards to keeping appropriate cell conditions within the whole microscopy chamber, including temperature, atmospheric composition of CO2 and humidity, and the pH and contents of the cell culture medium.

Matrix scans provide a local resolution of the signal throughout the well and thereby provide the opportunity to monitor seeding homogeneity and local variations of the transfection efficiency. In this measurement, 225 single measurement points were available, providing an image-like insight into the well and the level of transfection efficiency. Furthermore, this scan option allows exclusion of single measurement points as outliers. The spiral averaging measurement setting does not provide this resolution. However, it requires a fraction of the time (4 min spiral averaging vs. 34 min matrix scan per full 96-well plate) and provides data of comparable quality.

The results in fig. 2 and 3 confirm a linear relationship between the percentage of GFP+/mcherry+ HeLas  (= transfection efficiency) and the measured signal  for GFP or mcherry fluorescence with high accuracy  (R² = 0,9997 and 0,9998) and precision (%CV = 10.5 and 5.2).

Small fluctuations in temperature can cause issues with focusing the laser and microscope due to changes in the refractive index of the material, and constant evaporation from the cell culture flask only exacerbates this problem.

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Fluorescence is the process by which a photon is absorbed, and another of slightly lower energy, and therefore longer wavelength, is subsequently emitted. Under normal circumstances, the electrons of a fluorophore (a molecule capable of fluorescence) are in a low-energy ground state, which when excited by interaction with an incident photon may be promoted to a higher energy level.

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Following each of these steps, the primary or secondary antibody linked fluorophore can be added. In some cases, additional counter stains may be included to reduce background fluorescence.

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The laser utilized in confocal microscopy, besides illuminating a small section of the sample, can instead be used to excite fluorophores, massively improving the sensitivity of detection and signal-to-noise ratio when compared with reflected light from the sample alone.