The main observation method of optical microscope is fluorescence observation
Fluorescence phenomenon
Fluorescence refers to the process in which a fluorescent substance emits light with a longer wavelength almost simultaneously when irradiated with light of a specific wavelength (Figure 1). When light of a specific wavelength (excitation wavelength) strikes a molecule (such as a molecule in a fluorophore), the photon energy is absorbed by the molecule's electrons. Next, the electrons transition from the ground state (S0) to a higher energy level, the excited state (S1'). This process is called excitation①. The electron stays in the excited state for 10-9–10-8 seconds, during which the electron loses some energy ②. In the process of leaving the excited state (S1) and returning to the ground state ③, the remaining energy absorbed during the excitation process will be released.
The residence time of a fluorescent molecule in the excited state is the fluorescence lifetime, generally in nanoseconds, which is an inherent characteristic of the fluorescent molecule itself. The technology of imaging using fluorescence lifetime is called Fluorescence Lifetime Imaging (FLIM), which can perform more in-depth functional and accurate measurements in addition to fluorescence intensity imaging to obtain molecular conformation, intermolecular interactions, and molecular microenvironments. information that is difficult to obtain with conventional optical imaging.
Another important property of fluorescence is the Stokes shift, the difference in wavelength between the excitation and emission peaks (Figure 2). Usually the emission light wavelength is longer than the excitation light wavelength. This is because after the fluorescent substance is excited and before the photon is released, the electrons will lose a part of the energy through the relaxation process. Fluorescent substances with larger Stokes shifts are easier to observe under a fluorescence microscope.
Fluorescence Microscopes and Fluorescence Filter Blocks
Fluorescence microscopy is an optical microscope that uses fluorescence properties to observe and image, and is widely used in cell biology, neurobiology, botany, microbiology, pathology, genetics and other fields. Fluorescence imaging has the advantages of high sensitivity and high specificity, and is very suitable for the observation of the distribution of specific proteins, organelles, etc. in tissues and cells, the study of co-localization and interaction, and the tracking of life dynamic processes such as changes in ion concentration.
Most molecules in cells do not fluoresce, and in order to see them, they must be fluorescently labeled. There are many methods of fluorescent labeling, which can be directly labeled (such as using DAPI to label DNA), or immunostaining using the antigen-binding properties of antibodies, or the target protein can be labeled with fluorescent proteins (such as GFP, green fluorescent protein), or reversible binding. synthetic dyes (such as Fura-2), etc.
At present, the fluorescence microscope has become the standard imaging equipment of various laboratories and imaging platforms, and it is a good helper for our daily experiments. Fluorescence microscopes are mainly divided into three categories: upright fluorescence microscopes (suitable for sectioning), inverted fluorescence microscopes (suitable for living cells, taking into account sections), fluorescent stereoscopic microscopes (suitable for larger specimens, such as plants, zebrafish (adult/embryo) , medaka, mouse/rat organs, etc.).
The fluorescence filter block is the core component of fluorescence imaging in microscopes. It consists of an excitation filter, an emission filter and a dichroic beam splitter. It is installed in the filter wheel, such as the Leica DMi8 equipped with a 6-position filter wheel (Figure 3). ). Different microscopes have different wheel positions, and some microscopes use filter slides.
The filter block plays an important role in fluorescence imaging: the excitation filter selects the excitation light to excite the sample and blocks other wavelengths of light; the light passing through the excitation filter passes through a dichroic beamsplitter (its role is to reflect the excitation light and transmit the fluorescence), After reflection, it is focused by the objective lens, irradiated to the sample, and the corresponding fluorescence is excited to emit light. The emitted light is collected by the objective lens, passes through the dichroic beam splitter, and reaches the emission filter. As shown in Figure 4: the excitation wavelength is 450-490nm, the dichroic beam splitter reflects light shorter than 510nm, transmits light longer than 510nm, and the emission light receiving range is 520-560nm.
Fluorescence filters commonly used in fluorescence microscopes can be divided into two types: long pass (LP for short) and band pass (BP for short). The bandpass is usually determined by the central wavelength and the width of the interval, such as 480/40, which means that light from 460-500nm can be passed. Longpass filters, such as 515 LP, pass light with wavelengths longer than 515 nm (Figure 5).
Fluorescent substances have their characteristic excitation (absorption) curve and emission curve, the excitation peak is the ideal excitation wavelength (high excitation efficiency, which can reduce the excitation light energy, protect cells and dyes), and the emission curve is the emission fluorescence wavelength range. Therefore, in the experiment, we will choose the wavelength as close to the excitation peak as possible for excitation, and the receiving range needs to include the emission peak. For example, the excitation peak of Alexa Fluor 488 is 500nm, and the excitation filter of 480/40 can be selected in the fluorescence microscope.
Details of the filter cubes can be viewed in the microscope imaging software. Understanding dyes and finding the filter that best matches your sample is critical for fluorescence imaging. The spectral information of fluorescent dyes and fluorescent proteins is generally indicated in the instructions, and can also be found online.
In addition to the excitation and emission wavelengths of fluorescent probes, the selection of filter blocks also needs to consider whether there is non-specific excitation and whether there is cross color for multicolor labeled samples. In addition, it is necessary to consider the fluorescent light source used. At present, the commonly used fluorescent light sources include mercury lamps, metal halide lamps, and LED light sources that have developed rapidly in recent years. The spectrum of a fluorescent light source is continuous or discontinuous, and the energy will be different in different wavelength bands. LED light source is gradually becoming the main light source of fluorescence microscope due to its relatively narrow spectral band, more stable energy output, long life, safer and environmental protection and many other advantages.
In addition to the built-in filter block of the microscope, there is also an external fast wheel (Figure 7). The conversion speed of the filter adjacent to the external fast wheel of Leica is 27ms, which can realize high-speed multi-color experiments, such as FRET and Fura2 ratio calcium imaging. (Fig. 8) et al.
A wide variety of fluorescence microscopy imaging techniques
In order to meet different fluorescence imaging needs, in addition to fluorescence microscopes, various fluorescence microscopy imaging solutions have been developed:
Wide-field high-definition imaging systems, such as Leica THUNDER Imager, use Leica's innovative Clearing technology to efficiently remove non-focal plane interference signals during imaging, presenting clear images and the advantages of high-speed imaging;
The confocal laser scanning microscope uses pinholes to eliminate non-focal plane interference, realizes optical sectioning, and obtains high-definition images and three-dimensional images;
Ultra-high-resolution microscopes and nano-microscopes that break through the diffraction limit can observe fine structures smaller than 200 nm;
A multiphoton imaging system using the principle of multiphoton excitation for imaging of thick tissue and deep in vivo;
Light sheet imaging technology with high temporal and spatial resolution, fast imaging speed, high resolution, low phototoxicity, especially suitable for research on development and in vivo dynamic observation;
Fluorescence lifetime imaging (FLIM), which is not affected by factors such as fluorescent substance concentration, photobleaching, excitation light intensity, etc., can perform more in-depth functional and accurate measurements;
Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross Correlation Spectroscopy (FCCS), measure the molecular number and diffusion coefficient of fluorescent molecules, thereby analyzing molecular concentration, molecular size, viscosity, molecular motion, molecular binding/dissociation, and molecular optical properties;
Total Internal Reflection Fluorescence Microscopy (TIRF), with extremely high z-axis resolution, is ideal for studying the molecular structure and dynamics of cell membrane surfaces.
