Which Microscope to use

Confocal vs Wide Field Microscopy

Introduction

Wide field fluorescence microscopy is an imaging technique where fluorescent dyes or proteins in the specimen are excited at the same time trough an objective. Emitted fluorescence is captured by the same objective and is transmitted to a camera (or eyepieces).

In confocal fluorescence microscopy, light from a laser that passes a excitation pinhole is focused into the specimen to excite the fluorescent dyes or proteins. A pinhole allows the fluorescence signal, from the focus point only, to reach the detector. This strongly reduces the acquisition of out-of-focus light, providing optical sectioning and improving image quality. To get a confocal image the specimen has to be scanned point by point.

 

Optical Sectioning

The picture below shows the benefit of optical sectioning in confocal microscopy with a Thin and Thick specimen. You can see that optical sectioning is especially beneficial in thick samples.

 

Thick Specimen
Thick Specimen
Thick Specimen
Thin Specimen
Thin Specimen
Multi-color

Wide field microscope with more than one camera are rare. So taking multi-color (muli fluorescent-labels) images have to be acquired, color after color. This slows down imaging and fast processes in living-cells can have alignment problems. You also need a special filter set (dichroic+excitation/emission filter) for every single color/fluorescent-label. In most confocal you will find multiple detectors (in the Leica SP8 up to 5) and very often with spectral freedom (no filtersets needed). In the Leica SP8 a White light laser (WLL) is used in combination with fast variable (acousto optical beam splitter, AOBS) dichroic. Together with the spectral detector you can image up to 5 color at the same time.

Cons and Pros

Wide field:

  • Fast (one color) (Live-Cells, Dynamic processes, high-content/high-throughput screening)
  • Suitable for Thin Samples (cell culture, trans-well, thin sections)
  • Less noise compared to confocal (see below)

Confocal

  • Optical Sectioning (Thick samples, fine details, colocalization of colors)
  • Fast multi-color
  • Suitable for Thin and Thick (up to 200 µm) Samples (cell cultures, spheroids, organoids, thick sections, zebrafish and other species)
  • More noise compared to wide field (see below)
  • Functional imaging (FRAP, FRET, FLIM)
Noise

Although, compared to wide-field, confocal images quality can be higher, there is the fact that to acquire a confocal image you have to scan the specimen. In confocal microscopy exposure time per point(pixel) is typically less than a microsecond. For 4 million pixels (2048×2048) this results in a total of 4 seconds per image frame. In wide field microscopy a typical camera has 4 million pixels, so the exposure time per pixel is 4 million times longer with this same 4 seconds per image frame. To solve this problem, a confocal uses a laser with reasonable high power to get a high level of excitation light in a small focus point. However the concentration of fluorescent dyes or proteins in a specimen is limited and fluorophores have a so called life-time (it can take a few nanoseconds to go from an excited state back to the ground state, emitting a photon). This results, depending on the concentration of fluorescent dyes or proteins and their life-time, in capturing a limited number of photons in one microsecond exposure. Typical numbers ar 1-100 photons  in confocal microscopy where in wide field this is typically 1000-40,000 photons per pixel. As a consequence, imaging in confocal is slower and/or noise is higher. More about noise in fluorescent imaging see sidestep “Imaging, Noise and Quality“.