ABSTRACT
The use of automated techniques for image analysis of microscopic wood specimens together with new procedures for the preparation of stained xylem tissue support the use of quantitative wood anatomy. These techniques and procedures are especially useful in the studies of retrospective analysis of xylem phenology, reaction(s) of trees to stressful conditions of growth, or reconstruction of long-term growth trends. The unresolved technical problems during the digitalization of cross sections from entire increment cores were stabilization and precise shifting of long microscopic specimens onto the optical microscope stage. For this reason, we have developed a long slide holder for microscope stages in two versions: the basic one allowing stabilization and manual shifting, and the advanced one for stabilization and mechanical shifting. Both versions of the adapter speed up the work with long slides, improving the quality of panoramic images of microscopic specimens.
INTRODUCTION
Quantitative wood anatomy (QWA) involves a quantitative study on the temporal and spatial variability of anatomical features, mainly of the xylem of trees and shrubs in the context of its functional traits over time depending on environmental conditions (Fonti et al. 2010; von Arx et al. 2016). A dynamic development of microscope image analysis techniques that enable an automatic recognition and measurement of anatomical parameters, such as lumen area or cell wall thickness, allows to overcome the constraints associated with the time-consuming data collection on time-series of tracheid-lumen formation (von Arx et al. 2014). Concurrently, the invention of the core-microtome (Gärtner & Nievergelt 2010), a tool enabling the preparation of cross sections from entire increment cores, and the development of protocols for preparation techniques, allowed further progress in the field of sample preparation (Gärtner & Schweingruber 2013; Schneider & Gärtner 2013; Gärtner et al. 2015a, b; Ivanova et al. 2015). One of the key elements in the preparation of digital images of microscopic specimens is the digitization of their cross sections using a light microscope coupled to a digital camera (Gärtner et al. 2015a). This process is far less labour-intensive when using a digital slide scanner for the automated capturing of digital images from standard microscope slides as known from medical applications (Soenksen 2004; Isse et al. 2012). Unfortunately, long slides cannot be handled by any automated slide scanner. A first test for an advanced system handling long slides failed (H. Gärtner, personal communication). Consequently, in case of using the new long micro slides comprising entire increment cores, one would be restricted to the use of common transmitted light microscopes. These microscopes are equipped with holders for standard slides of 76 × 25 mm. So far, the problem of stabilizing long slides on the microscope stage during the digitization process has not yet been solved satisfactorily. Until today, a simple cardboard adapter mounted on the cross table instead of a conventional microscope slides holder is the only solution available (Gärtner et al. 2015b ). This rather basic adapter allows the microscope slide to be maintained in a position perpendicular to the axis of the microscope lens only while operating in the middle of the slide. When the slide is moved to extreme positions, the lack of support along the entire length of the specimen causes its destabilization. Moreover, the ability to use the micro-screws of the microscope stage for a precise shift of the specimen is limited by their maximum range of motion in the x-axis, usually around 80 mm. Therefore, shifting a 400 mm long specimen on the microscope stage is performed manually with limited precision (Ivanova et al. 2015).
Here, we present a high-precision adapter designed for long microscope slides in order to solve both of the above-mentioned problems, namely stabilizing and precise shifting long specimens on the microscope stage. These limitations were overcome by creating two new slide adapters: the basic version allowing stabilization and manual slide shift, and the advanced version allowing mechanical shift using a dedicated micro-screw.
Long slide holder with manual shift
The basic adapter allows the stabilization of long (400 mm) microscopic slides and their shifting either using micro-screws of the microscope stage or simply manually. The adapter design is a 740 mm long and 106 mm wide plastic base stabilized with aluminium profiles (Fig. 1). This construction is fixed to the microscope stage through an aluminium profile designed in such a way that it enables attachment to various microscope types (e.g. Olympus, Zeiss) (Fig. 3A). The adapter base is shielded from above with a transparent plastic mold creating a free space for the placement of microscopic specimens with the maximum thickness of 3 mm and width of 35 mm. Fixing of the slide position in the adapter is performed by means of four eccentric screws with rubber O-rings spaced along the aluminium profile at 182 mm intervals. The adapter can be adjusted to hold wider microscopic slides (up to 40 mm) by removing a separate plastic spacer. The upper, transparent part of the adapter has a rectangular window of 100 × 39 mm allowing for microscopic observation, and longitudinal slots allowing for direct manual shift of the specimen.
Scheme of long slide holder with manual shifting. Dashed arrows indicate place of fixing long slide. The labels with arrows indicate the key elements of the construction: four eccentric screws with the rubber O-rings, mounting plate for fixing to the microscope stage, window for microscopic observation, plastic spacer described in the text.
Citation: IAWA Journal 39, 4 (2018) ; 10.1163/22941932-20170213
Scheme of long slide holder with mechanical shifting system. Dashed arrows indicate place of fixing long slide. The labels with arrows indicate the key elements of the construction: C-shaped handles, clamping screw, clutch with a felt spindle, micro-screw for slide shifting, mounting plate for fixing to the microscope stage, window for microscopic observation described in the text.
Citation: IAWA Journal 39, 4 (2018) ; 10.1163/22941932-20170213
Long slide holder with mechanical shifting system
An advanced version of the adapter, besides the stabilization of long (400 mm) microscopic slides, allows for mechanical shifting of the specimens using a dedicated micro-screw. The construction of this adapter is significantly different from that of the basic version. The base is made up of two non-transparent plastic plates of 112 × 98 mm, framed by a closed aluminium profile. The adapter is fixed to the microscope stage with an aluminium plate profiled as in the basic version (Fig. 2).
The 400 mm glass slide is stabilized by two C-shaped handles, which at the same time allow the adjustment of their spacing, thus fitting them to the precise length and width of the slide with two screws (Fig. 3B). The maximum width of the slide that can be fixed in the holders is 40 mm. The microscopic slide movement along the adapter is carried out along the guide, which simultaneously stabilizes one of the edges of the adapter. Slide shifting is controlled with a micro-screw located on the right side of the guide. Rotation of the micro-screw on the holder is controlled by a clutch with a felt spindle. The pin of the felt spindle to micro-screw is adjustable via the clamping screw. Release of the clutch screw allows for manual, rapid slide shifting along the entire guide (Fig. 4).
Long slide holders mounted on a microscope stage - Olympus BX 50. – Panel A: long slide holder with manual shifting. The labels with arrows indicate the four eccentric screws with the rubber O-rings described in the text. – Panel B: long slide holder with mechanical shifting. The labels with arrows indicate the key elements of the construction: C-shaped handles, clamping screw, clutch with a felt spindle, micro-screw for slide shifting.
Citation: IAWA Journal 39, 4 (2018) ; 10.1163/22941932-20170213
Long slide holder with adjustable spacing. – Panel A: slide length adjustment. – Panel B: slide width adjustment. The labels with arrows indicate C-shaped handles described in the text.
Citation: IAWA Journal 39, 4 (2018) ; 10.1163/22941932-20170213
Possible applications
The use of automated techniques for the analysis of xylem microscope slide images allows to investigate the intra-annual growth pattern of trees in response to seasonal variability (Castagneri et al. 2017; Prendin et al. 2017). Quantitative analysis of the structural and functional parameters of xylem anatomical elements allows for the construction of cell parameter chronologies, enabling a retrospective analysis of xylem phenology, and indirectly the activity of vascular cambium in the context of key climatic parameters (Carrer et al. 2017). Extreme climatic events that are too short to allow a clear trace in proxies commonly used in dendroclimatology such as ring width, ring density, or even maximum latewood density can be identified in tree-ring anatomical profiles (Carrer et al. 2016). This applies to both the anatomical structure of gymnosperms and to the much more complex anatomical structure of angiosperms (Fonti et al. 2009; Wegner et al. 2013; von Arx et al. 2015; Petit et al. 2016; Prendin et al. 2017). In ring-porous species quantitative analysis of anatomical structures allows to study specific features such as earlywood vessel grouping or potentially the diameter of latewood vessels (Fonti et al. 2013). The potential for QWA time-series applications is not limited to tracking the growth pattern in the aboveground part of the plant; this method can also be successfully used to study anatomical variations in the tree roots over longer time periods (von Arx et al. 2005; Wrońska-Wałach et al. 2016). The above-mentioned applications of microscopic image analysis techniques in xylem studies may require stabilization of long specimens on the microscope stage during their digitization process, especially when cross sections with entire increment cores are used. The proposed devices allow proper stabilization of long microscopic slides as well as a precise handling of the slides during image processing, which makes the whole process much easier.
Nevertheless, as long as the process of digitization of long micro-sections is not fully automated, expanding these types of applications onto multi-species and multi-environmental studies still encounters a labour intensity barrier. Therefore, future work on the development of the long slide holder for microscope stages should be focused on designing a motor-driven and software controlled capturing process.
According to our experience, the time needed for digitizing a long section along a line restricted to the width of a single image at 100× magnification (approx. 1 mm), is about 15–30 minutes, depending on the overlap needed between single images. When digitizing an entire long anatomical slide (total length and width), it might take up to 2–4 hours at 100 × magnification. However, it may be extended due to the width of the micro-sections, the software used to operate the microscope camera (Z-Stack technique), homogeneity of the anatomical structure affecting the necessity to increase the overlapping area of adjacent images. When the quality of micro-slides does not allow for uniform focusing due to buckling, the solution can be the Z-Stack technique enabling combination of the focused image information from multiple images (von Arx et al. 2016). As a result, this capturing procedure allows to obtain high-resolution images at the same time; however, it significantly increases the labour-intensity of the digitization process.
Both types of long slide stabilizers can be ordered at TECH-FORM Sp. z o. o. Company (Poland), which is currently working on a more advanced version that takes into account the user experience. The main improvements relate to the construction of left- and right-hand micro-screw and the increase of the speed of the slide shift.
ACKNOWLEDGMENTS
We thank Roman Mańka and Adrian Holek from TECH-FORM Sp. z o.o. Company for constructing and further developing the long slide adapter. This research was performed under the Forest Research Institute statutory aid No. 24.02.38 of the Ministry of Science and Higher Education in Poland. This research is linked to activities conducted within the COST FP1106 “STReESS” network. This paper was developed under a granted Scholarship within Scholarship Fund of the Forest Research Institute, pursuant to the decision of the Head of the Institute dated 5th October, 2016 (based on an Agreement concluded on 6th October, 2016).
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Corresponding author: e-mail: m.klisz@ibles.waw.pl
