9 Form Test Plate (version 1.0) by Diatom Lab
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1 9 Form Test Plate (version 1.0) by Diatom Lab Instruction Manual (Copyright 2017 Diatom Lab, Diatom Shop, Stefano Barone)
2 PREMISE: A) This microscope slide is a Diatom RESOLUTION TEST containing 9 (cleaned, selected and micromanipulated in Diatom Lab's laboratory) Diatom species with striae, areolae and lineolae that can be resolved using a light microscope. The resolving power of a microscope is measured by its ability to differentiate two lines or points in an object. This microscope slide can be also used as a TRAINING TOOL to work with light microscopes, or as a TEACHING TOOL: for example, it is possible to - examine the variations in contrast and resolution by regulating the condenser aperture diaphragm; - understand the importance of correction collar for minimazing spherical aberration; - examine the variations in resolution by using different wavelengths of light B) It's a STANDARDIZED TEST, in fact every microscope slide has the same production characteristics: - In order to ensure maximum resolution Diatom frustules are internally mounted on the lower surface of the cover glass (and not on microscope slide!) which has a thickness of 0,17 mm: although this procedure requires special manufacturing precautions, it ensures the best way to observe Diatoms, in fact medium and high power microscope objectives are often manufactured to offer the best results with a cover glass thickness of 0,17 mm. Furthermore, mounting on the internal surface of cover glass permits to easily use oil immersion objectives too (even those with very short free working distance, such as 63x/1,4 or 100x/1,4) as there is no space between diatoms and cover glass! - the customized cover glass with high optical quality has been manufactured in Germany specifically for Diatom Lab; - the mountant (Diatom Cubed, produced in Diatom Lab) has a high refractive index and belongs to the same production lot; - each species belongs to a sample collected in the same place / depth / time C) Attached images refer to the live-view window of the camera (they are real time images, no focus stacking technique has been used) D) These tests have been made using a Zeiss Axio Imager.A2 research microscope, equipped for the occasion with the following accessories: dry and oil-immersion infinity-corrected Plan-Apochromat and EC Plan Neofluar objectives; dry (0,9) and oil-immersion (1,4) condensers; Zeiss VIS-LED (5600K). In the present case NO BOOSTERS (such as blue color filters, oblique illumination and UV illumination) have been used (UV illumination is dangerous for the user in absence of special precautions), but in some cases color filters and oblique illumination are highly recommended to the user in absence of Differential interference contrast and oil-immersion darkfield condensers. E) Not achieving similar result doesn't always mean the user has poor quality microscopes, objectives and accessories: besides numerical aperture of microscope objectives, also the microscopist's experience is important. Finally, it is useful to verify the microscope alignment and the cleanliness of the optics following the microscope instruction manual. Note well: the 9 Form Test Plate and other microscope slides can be ordered by (info@diatomshop.com) Happy microscope observations by Diatom Lab! Stefano Barone THE FOLLOWING PAGES CONTAIN THE TEST INSTRUCTIONS
3 Diatom Number 1 Species: Cymbella mexicana Recommended microscope objectives: 10x (at most 20x) Details to resolve: areolae inside the striae must appear sharp and well-spaced Suggested techniques to improve resolution: oblique illumination or Differential interference contrast Diatom Number 2 Species: Epithemia turgida Recommended microscope objectives: 10x and 20x Suggested techniques to improve resolution: in case oblique illumination or Differential interference contrast Diatom Number 3 Species: Anomoeoneis polygramma Recommended microscope objective: 20x Suggested techniques to improve resolution: in case oblique illumination, Dark Field and Differential interference contrast
4 Diatom Number 4 Species: Triceratium favus. (curiosity: this species was used as test in the Nineteenth century) Recommended microscope objective: 20x Details to resolve: pores inside the hexagonal chambers must appear clearly visible Suggested techniques to improve resolution: oblique illumination, Dark Field, Differential interference contrast, Phase Contrast Diatom Number 5 Species: Eunotia pectinalis (connective view) Recommended microscope objectives: 20x with a high numerical aperture or (dry, oil-immersion) 40x Details to resolve: areolae in the central area must appear sharp Suggested techniques to improve resolution: 20x objective with Differential interference contrast, or 40x objective without oblique illumination Diatom Number 6 Species: Cocconeis placentula (epivalve and hypovalve are clearly different) Recommended microscope objectives: dry or oil-immersion 40x Suggested techniques to improve resolution: Differential interference contrast and Dark Field in case of dry objectives; Bright Field (without oblique illumination) in case of oil-immersion 40x objectives
5 Diatom number 7 Species: Diploneis smithii Recommended microscope objective: oil-immersion 40x objective Suggested techniques to improve resolution: oblique illumination and Differential interference contrast. Similar results can be achieved using Differential interference contrast with a high numerical aperture dry 40x objective Diatom Number 8 Species: Surirella striatula (curiosity: frustule has a torsion along the apical axis) Recommended microscope objectives: oil-immersion 63 or 100x objectives having an excellent numerical aperture (1,3 or better 1,4) with immersion condenser Details to resolve: areolae inside the striae must appear sharp and well-spaced (striae are a test item too, as can be resolved using a good 20x objective with oblique illumination or Differential interference contrast) Suggested techniques to improve resolution: Differential interference contrast; Dark field illumination using an immersion 1,2/1,4 dark field condenser (and therefore oil immersion objectives); strong oblique illumination with an immersion condenser and blue filter Diatom Number 9 Species: Navicula oblonga. In Diatom Lab's sample the AVERAGE DISTANCE BETWEEN PORES (lineolae) of this species is 0,14 µm (micrometers or microns), see Scanning Electron Microscope (SEM) measurements in front page. The limit of resolution of most light microscopes is 0.2 μm, but it can be bypassed by the techniques below. Recommended microscope objectives: oil-immersion 63 or 100x objectives having a good or excellent numerical aperture (from 1,2; better: 1,3 or 1,4) with immersion condenser Details to resolve: lineolae inside the striae Suggested techniques to improve resolution: Dark field illumination using an immersion dark field condenser (and therefore oil immersion objectives); Circular oblique illumination (C.O.L.) with polarized light in double immersion (immersion objective + immersion condenser);
6 UV illumination: in this case highly specialized laboratory facilities are required (it is dangerous for the eyes, it requires the use of special protection devices, accessories and cameras. Please refer to the operating manual of your instruments); Some variants of the Differential interference contrast (such as AVEC-DIC, the Allen Video-enhanced Contrast); IRC (Interference reflection contrast technique)
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