Confocal microscopy

Chapter focusing on confocal microscopy. A confocal microscope is one in which the illumination is confined to a small volume in the specimen, the detection is confined to the same volume and the image is built up by scanning this volume over the specimen, either by moving the beam of light over the specimen or by displacing the specimen relative to a stationary beam. The chief advantage of this type of microscope is that it gives a greatly enhanced discrimination of depth relative to conventional microscopes. Commercial systems appeared in the 1980s and, despite their high cost, the world market for them is probably between 500 and 1000 instruments per annum, mainly because of their use in biomedical research in conjunction with fluorescent labelling methods. There are many books and review articles on this subject ( e.g. Pawley ( 2006) , Matsumoto( 2002), Wilson (1990) ). The purpose of this chapter is to provide an introduction to optical and engineering aspects that may be o f interest to biomedical users of confocal microscopy

Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells

Standing-wave excitation of fluorescence is highly desirable in optical microscopy because it improves the axial resolution. We demonstrate here that multiplanar excitation of fluorescence by a standing wave can be produced in a single-spot laser scanning microscope by placing a plane reflector close to the specimen. We report that the relative intensities in each plane of excitation depend on the Stokes shift of the fluorochrome. We show by the use of dyes specific for the cell membrane how standing-wave excitation can be exploited to generate precise contour maps of the surface membrane of red blood cells, with an axial resolution of ~90 nm. The method, which requires only the addition of a plane mirror to an existing confocal laser scanning microscope, may well prove useful in studying diseases which involve the red cell membrane, such as malaria.Comment: 15 pages, 4 figures; changed the discussion of narrow-band detected fringes (Fig. 3) to describe the phenomenon as a moire pattern between the excitation and emission standing-wave fields, rather than a beats pattern; added DiI(5)-labelled red blood cell in Fig. 4 to show that standing-wave fringes are present even when the dye excitation wavelength is outside the haemoglobin absorption ban

Characterisation of a deep-ultraviolet light-emitting diode emission pattern via fluorescence

Recent advances in LED technology have allowed the development of high-brightness deep-UV LEDs with potential applications in water purification, gas sensing and as excitation sources in fluorescence microscopy. The emission pattern of an LED is the angular distribution of emission intensity and can be mathematically modelled or measured using a camera, although a general model is difficult to obtain and most CMOS and CCD cameras have low sensitivity in the deep-UV. We report a fluorescence-based method to determine the emission pattern of a deep-UV LED, achieved by converting 280 nm radiation into visible light via fluorescence such that it can be detected by a standard CMOS camera. We find that the emission pattern of the LED is consistent with the Lambertian trend typically obtained in planar LED packages to an accuracy of 99.6%. We also demonstrate the ability of the technique to distinguish between LED packaging types

Electron microscopic measurement of the size of the optical focus in laser scanning microscopy

We describe a method for measuring the lateral focal spot size of a multiphoton laser scanning microscope (LSM) with unprecedented accuracy. A specimen consisting of an aluminum film deposited on a glass coverslip was brought into focus in a LSM and the laser intensity was then increased enough to perform nanoablation of the metal film. This process leaves a permanent trace of the raster path usually taken by the beam during the acquisition of an optical image. A scanning electron microscope (SEM) was then used to determine the nanoablated line width to high accuracy, from which the lateral spot size and hence resolution of the LSM can be determined. To demonstrate our method, we performed analysis of a multiphoton LSM at various infrared wavelengths, and we report measurements of optical lateral spot size with an accuracy of 20 nm, limited only by the resolution of the SEM

Femtosecond synchronously in-well pumped vertical-external-cavity surface-emitting laser

We demonstrate the first synchronously in-well pumped vertical-external-cavity surface-emitting laser (VECSEL). Depending on the cavity mismatch, laser pulses with a duration from 1 ps to 7 ps at a repetition rate of 76 MHz were generated directly from the laser at 860 nm. The application of extra-cavity pulse compression further shortened the pulse to a duration of 210 fs providing a peak power of 226 W

Standing wave microscopy of red blood cell membrane morphology with high temporal resolution

Widefield fluorescence microscopy is an integral tool for life science imaging though the achievable resolutions are limited by the diffraction nature of light. One technique to increase the axial resolution is known as standing wave microscopy [1]. The standing wave can be generated by placing a mirror at the specimen plane which causes interference between the incoming and reflected excitation illumination. The axial resolution is reduced to λ/4n as only fluorophores which are in the location of the full width at the half maximum of the antinodes are excited [2] resulting in periodic bands of fluorescence

Multi-wavelength, all-solid-state, continuous wave mode locked picosecond Raman laser

We demonstrate the operation of a cascaded continuous wave (CW) mode-locked Raman oscillator. The output pulses were compressed from 28 ps at 532 nm down to 6.5 ps at 559 nm (first Stokes) and 5.5 ps at 589 nm (second Stokes). The maximum output was 2.5 W at 559 nm and 1.4 W at 589 nm with slope efficiencies up to 52%. This technique allows simple and efficient generation of short-pulse radiation to the cascaded Stokes wavelengths, extending the mode-locked operation of Raman lasers to a wider range of visible wavelengths between 500 - 650 nm based on standard inexpensive picosecond Nd:YAG oscillators